NOTCH 1 SPECIFIC siRNA MOLECULE

ABSTRACT

The present invention is related to a nucleic acid molecule comprising a double-stranded structure, wherein the double-stranded structure is formed by a first strand and a second strand, wherein the first strand consist of the following nucleotide sequence 5′ acGaGcUgGaCcAcUgGuCdT s dT 3′, and the second strand consists of the following nucleotide sequence 5′ GAcCaGuGgUcCaGcUcGudT s dT 3′, wherein a minor nucleotide indicates that the nucleotide is 2′-F modified and an underlined nucleotide indicates that the nucleotide is 2′-0-methyl modified and wherein dT s dT indicates that at the 3′ end a dinucleotide is attached consisting of two dT nucleotides, wherein said two dTs are covalently linked through a phosphorothioate bond.

The present invention is related to a nucleic acid molecule comprising adouble-stranded structure, the nucleic acid molecule comprising adouble-stranded structure for use in a method for the treatment and/orprevention of a disease, the nucleic acid molecule comprising adouble-stranded structure for use in a method of restoring drugsensitivity of cancer cells, use of nucleic acid molecule comprising adouble-stranded structure for the manufacture of a medicament, use ofthe nucleic acid molecule comprising a double-stranded structure in themanufacture of an agent for restoring drug sensitivity of cancer cells,a nanoemulsion comprising the nucleic acid molecule comprising adouble-stranded structure, the nanoemulsion for use in a method for thetreatment and/or prevention of a disease, the nanoemulsion for use in amethod of restoring drug sensitivity of cancer cells, use of thenanoemulsion structure for the manufacture of a medicament, use of thenanoemulsion in the manufacture of an agent for restoring drugsensitivity of cancer cells, a pharmaceutical composition comprising thenucleic acid molecule comprising a double-stranded structure, thepharmaceutical composition for use in a method for the treatment and/orprevention of a disease, the pharmaceutical composition for use in amethod for restoring drug sensitivity of cancer cells, a method for thetreatment and/or prevention of a disease comprising the administrationof the nucleic acid molecule comprising a double-stranded structure, amethod for restoring drug sensitivity of cancer cells comprising theadministration of the nucleic acid molecule comprising a double-strandedstructure, a kit comprising the nucleic acid molecule comprising adouble-stranded structure, the kit for use in a method of treatmentand/or prevention of a disease, the kit for use in a method forrestoring drug sensitivity of cancer cells, a kit comprising thenanoemulsion, the kit for use in a method of treatment and/or preventionof a disease, and the kit for use in a method for restoring drugsensitivity of cancer cells.

Notch 1 is a gene coding for a single-pass transmembrane receptor whichis also categorized as a Type 1 transmembrane protein. Human Notch 1 wasdescribed for the first time by Ellisen L W et al. (Ellisen L W et al.,Cell 66(4), 649-661 (1991)). Notch 1 is a member of the Notch family.Members of this family share structural characteristics including anextracellular domain consisting of multiple epidermal growth factor-like(EGF) repeats, and an intracellular domain consisting of multiple,different domain types. Notch family members play a role in a variety ofdevelopmental processes by controlling cell fate decisions. The Notchsignaling network is an evolutionarily conserved intercellular signalingpathway that regulates interactions between physically adjacent cells.Notch 1 and its translation product constitute a drugable target in manytumor entities. Sequence information including the nucleotide sequenceof the cDNA of human Notch 1 may, for example, be retrieved from GenBankentry NM_017617.3.

There is an ongoing need in the art for means of silencing or knockingdown the expression levels of Notch 1 in vitro and in vivo, includingthe use of siRNA for the treatment of disease which can be treated orprevented by decreasing the expression of the Notch 1 gene and morespecifically by decreasing the translation of mRNA coding for Notch 1.One group of diseases which can be treated that way are various tumordiseases and cancer.

Therefore, the problem underlying the present invention is the provisionof a means for silencing of knocking down Notch 1 and more preferablysilencing or knocking down the expression levels of Notch 1 in vitro andin vivo. A further problem underlying the invention is the provision ofa method for the treatment of a disease, more preferably disease whichcan be treated or prevented by decreasing the expression of the Notch 1gene and more specifically by decreasing the translation of mRNA codingfor Notch 1, and of a means which is useful in such method. A stillfurther problem underlying the problem is the provision of a method forrestoring drug sensitivity of cancer cells, and of a means which isuseful in such method. Finally, the problem underlying the presentinvention is the provision of a method for adjunct therapy in thetreatment of cancer, and of a means which is useful in such method.

These and other problems underlying the present invention are solved bythe subject matter of the attached independent claims. Preferredembodiments may be taken from the attached dependent claims.

Certain embodiments will become apparent to the skilled person in viewof the description, including those embodiments set forth below. Thoseembodiments set forth below equally solve the above and other problemsunderlying the present invention.

EMBODIMENT 1

A nucleic acid molecule comprising a double-stranded structure,

wherein the double-stranded structure is formed by a first strand and asecond strand,wherein the first strand comprises a first stretch of contiguousnucleotides and the second strand comprises a second stretch ofcontiguous nucleotides,wherein the first stretch of contiguous nucleotides comprisesa) a nucleotide sequence, wherein the nucleotide sequence is at least63% identical to

(i) nucleotide sequence (SEQ ID NO: 1) 5′ ACGAGCUGGACCACUGGUC 3′ or (ii)nucleotide sequence (SEQ ID NO: 8) 5′ CGAGCUGGACCACUGGU 3′;orb) a nucleotide sequence, wherein the nucleotide sequence comprises atleast a stretch of 8 or 9 nucleotides of

(i) nucleotide sequence (SEQ ID NO: 1) 5′ ACGAGCUGGACCACUGGUC 3′ or (ii)nucleotide sequence (SEQ ID NO: 8) 5′ CGAGCUGGACCACUGGU 3′andwherein the nucleic acid molecule is capable of causingpost-transcriptional silencing of a gene.

EMBODIMENT 2

The nucleic acid molecule of embodiment 1, wherein post-transcriptionalsilencing of a gene is RNA interference.

EMBODIMENT 3

The nucleic acid molecule of any one of embodiments 1 to 2, wherein thegene is Notch 1, preferably human Notch 1.

EMBODIMENT 4

The nucleic acid molecule of any one of embodiments 1 to 3, wherein thenucleic acid molecule is capable of degrading mRNA coding for the geneor a precursor of said mRNA, preferably in a cell.

EMBODIMENT 5

The nucleic acid molecule of embodiment 4, wherein the nucleotidesequence of a cDNA of the mRNA is available from GenBank entryNM_017617.3.

EMBODIMENT 6

The nucleic acid molecule of any one of embodiments 1 to 5, wherein thecDNA consists of the nucleotide sequence of SEQ ID NO: 2

EMBODIMENT 7

The nucleic acid molecule of any one of embodiments 1 to 6, wherein thesecond stretch of contiguous nucleotides is at least partiallycomplementary to a part of the first stretch of contiguous nucleotides.

EMBODIMENT 8

The nucleic acid molecule of any one of embodiments 1 to 7, wherein thesecond stretch of contiguous nucleotides is at least partiallycomplementary to the first stretch of contiguous nucleotides.

EMBODIMENT 9

The nucleic acid molecule of any one of embodiments 1 to 8, wherein thefirst stretch of contiguous nucleotides comprises 13 to 29 nucleotides,preferably 17 to 25 or 19 to 25 nucleotides and more preferably 19 to 23nucleotides.

EMBODIMENT 10

The nucleic acid molecule of any one of embodiments 1 to 9, wherein thesecond stretch of contiguous nucleotides comprises 13 to 29 nucleotides,preferably 17 to 25 or 19 to 25 nucleotides and more preferably 19 to 23nucleotides.

EMBODIMENT 11

The nucleic acid molecule of any one of embodiments 1 to 10, wherein thefirst stretch of contiguous nucleotides and the second stretch ofcontiguous nucleotides comprises 13 to 29 nucleotides, preferably 17 to25 or 19 to 25 nucleotides and more preferably 19 to 23 nucleotides.

EMBODIMENT 12

The nucleic acid molecule of any one of embodiments 9 to 11, wherein thenucleotides are consecutive nucleotides.

EMBODIMENT 13

The nucleic acid molecule of any one of embodiments 1 to 12, wherein thefirst strand consists of the first stretch of contiguous nucleotides.

EMBODIMENT 14

The nucleic acid molecule of any one of embodiments 1 to 13, wherein thesecond strand consist of the second stretch of contiguous nucleotides.

EMBODIMENT 15

The nucleic acid molecule of any one of embodiments 1 to 14, wherein thefirst strand consists of the first stretch of contiguous nucleotides andthe second strand consists of the second stretch of contiguousnucleotides.

EMBODIMENT 16

The nucleic acid molecule of any one of embodiments 1 to 14, wherein thedouble-stranded structure comprises 13 to 29 base pairs, preferably 16to 27 or 19 to 25 base pairs and more preferably 19 to 23 base pairs.

EMBODIMENT 17

The nucleic acid molecule of any one of embodiments 1 to 16, wherein thefirst stretch of contiguous nucleotides comprises

(i) nucleotide sequence (SEQ ID NO: 1) 5′ ACGAGCUGGACCACUGGUC 3′ or (ii)nucleotide sequence (SEQ ID NO: 8) 5′ CGAGCUGGACCACUGGU 3′.

EMBODIMENT 18

The nucleic acid molecule of any one of embodiments 1 to 17, wherein thesecond stretch of contiguous nucleotides comprises

(i) nucleotide sequence (SEQ ID NO: 3) 5′ GACCAGUGGUCCAGCUCGU 3′ or (ii)nucleotide sequence (SEQ ID NO: 9) 5′ ACCAGUGGUCCAGCUCG 3′.

EMBODIMENT 19

The nucleic acid molecule of any one of embodiments 17 to 18, whereinthe first stretch of contiguous nucleotides comprises

(i) nucleotide sequence (SEQ ID NO: 1) 5′ ACGAGCUGGACCACUGGUC 3′ or (ii)nucleotide sequence (SEQ ID NO: 8) 5′ CGAGCUGGACCACUGGU 3′,andthe second stretch of contiguous nucleotides comprises

(i) nucleotide sequence (SEQ ID NO: 3) 5′ GACCAGUGGUCCAGCUCGU 3′ or (ii)nucleotide sequence (SEQ ID NO: 9) 5′ ACCAGUGGUCCAGCUCG 3′.

EMBODIMENT 20

The nucleic acid molecule of any one of embodiments 1 to 19, wherein thenucleic acid molecule is blunt ended at at least one end.

EMBODIMENT 21

The nucleic acid molecule of embodiment 20, wherein the nucleic acidmolecule is blunt ended at the end defined by the 5′ end of the firststrand and the 3′ end of the second strand.

EMBODIMENT 22

The nucleic acid molecule of embodiment 20, wherein the nucleic acidmolecule is blunt ended at the end defined by the 3′ end of the firststrand and the 5′ end of the second strand.

EMBODIMENT 23

The nucleic acid molecule of any one of embodiments 20 to 22, whereinthe nucleic acid molecule is blunt ended at the end defined by the 5′end of the first strand and the 3′ end of the second strand and at theend defined by the 3′ end of the first strand and the 5′ end of thesecond strand.

EMBODIMENT 24

The nucleic acid molecule of any one of embodiments 1 to 19, wherein thenucleic acid molecule has an overhang at at least one end.

EMBODIMENT 25

The nucleic acid molecule of embodiment 24, wherein the nucleic acidmolecule has an overhang at the end defined by the 5′ end of the firststrand and the 3′ end of the second strand.

EMBODIMENT 26

The nucleic acid molecule of embodiment 25, wherein the overhang is a 5′overhang.

EMBODIMENT 27

The nucleic acid molecule of embodiment 25, wherein the overhang is a 3′overhang.

EMBODIMENT 28

The nucleic acid molecule of embodiment 24, wherein the nucleic acidmolecule has an overhang at the end defined by the 3′ end of the firststrand and the 5′ end of the second strand.

EMBODIMENT 29

The nucleic acid molecule of embodiment 28, wherein the overhang is a 5′overhang.

EMBODIMENT 30

The nucleic acid molecule of embodiment 28, wherein the overhang is a 3′overhang.

EMBODIMENT 31

The nucleic acid molecule of embodiment 24, wherein the nucleic acidmolecule has an overhang at the end defined by the 5′ end of the firststrand and the 3′ end of the second strand and at the end defined by the3′ end of the first strand and the 5′ end of the second strand.

EMBODIMENT 32

The nucleic acid molecule of embodiment 31, wherein the overhang is a 5′overhang.

EMBODIMENT 33

The nucleic acid molecule of embodiment 32, wherein the overhang is a 3′overhang.

EMBODIMENT 34

The nucleic acid molecule of any one of embodiments 24 to 33, whereinthe overhang consists of one, two, three, four or five nucleotides.

EMBODIMENT 35

The nucleic acid molecule of embodiment 34, wherein the overhangconsists of two nucleotides.

EMBODIMENT 36

The nucleic acid molecule of any one of embodiments 34 to 35, whereinthe nucleotide is dT.

EMBODIMENT 37

The nucleic acid molecule of any one of embodiments 1 to 19 and 24 to36, wherein the first stretch of contiguous nucleotides comprises

(i) nucleotide sequence (SEQ ID NO: 4) 5′ ACGAGCUGGACCACUGGUCdTdT 3′ or(ii) nucleotide sequence (SEQ ID NO: 10) 5′ CGAGCUGGACCACUGGUdTdT 3′.

EMBODIMENT 38

The nucleic acid molecule of any one of embodiments 1 to 19 and 24 to37, wherein the second stretch of contiguous nucleotides comprises

(i) nucleotide sequence (SEQ ID NO: 5) 5′ GACCAGUGGUCCAGCUCGUdTdT 3′ or(ii) nucleotide sequence (SEQ ID NO: 11) 5′ ACCAGUGGUCCAGCUCGdTdT 3′.

EMBODIMENT 39

The nucleic acid molecule of any one of embodiments 37 to 38, whereinthe first stretch of contiguous nucleotides comprises

(i) nucleotide sequence (SEQ ID NO: 4) 5′ ACGAGCUGGACCACUGGUCdTdT 3′ or(i) nucleotide sequence (SEQ ID NO: 10) 5′ CGAGCUGGACCACUGGUdTdT 3′,and the second stretch of contiguous nucleotides comprises

(i) nucleotide sequence (SEQ ID NO: 5) 5′ GACCAGUGGUCCAGCUCGUdTdT 3′ or(ii) nucleotide sequence (SEQ ID NO: 11) 5′ ACCAGUGGUCCAGCUCGdTdT 3′.

EMBODIMENT 40

The nucleic acid molecule of any one of the preceding embodiments,wherein the first strand and the second strand are covalently linked toeach other, preferably the 3′ end of the first strand is covalentlylinked to the 5′ end of the second strand.

EMBODIMENT 41

The nucleic acid molecule of any one of embodiments 1 to 40, wherein oneor more of the nucleotides forming the first stretch of contiguousnucleotides is modified.

EMBODIMENT 42

The nucleic acid molecule of any one of embodiments 1 to 41, wherein oneor more of the nucleotides forming the second stretch of contiguousnucleotides is modified.

EMBODIMENT 43

The nucleic acid molecule of any one of embodiments 41 and 42, whereinone or more of the nucleotides forming the first stretch of contiguousnucleotides is modified and one or more of the nucleotides forming thesecond stretch of contiguous nucleotides is modified

EMBODIMENT 44

The nucleic acid molecule of any one of embodiments 1 to 43, wherein oneor more of the nucleotides forming the first strand is modified.

EMBODIMENT 45

The nucleic acid molecule of any one of embodiments 1 to 44, wherein oneor more of the nucleotides forming the second strand is modified.

EMBODIMENT 46

The nucleic acid molecule of any one of embodiments 44 to 45, whereinone or more of the nucleotides forming the first strand is modified andone or more of the nucleotides forming the second strand is modified.

EMBODIMENT 47

The nucleic acid molecule of any one of embodiments 41 to 46, whereinthe modification of the one or more of the nucleotides is a modificationof the sugar moiety of the one or more nucleotides and/or a modificationof the phosphate moiety of the one or more nucleotides.

EMBODIMENT 48

The nucleic acid molecule of embodiment 47, wherein the modification ofthe sugar moiety is selected from the group comprising 2′O-methyl and2′-F.

EMBODIMENT 49

The nucleic acid molecule any one of embodiments 47 to 48, wherein themodification of the phosphate moiety is such that a phosphorothioatelinkage is formed between two nucleotides.

EMBODIMENT 50

The nucleic acid molecule of any one embodiments 41 to 43 and 47 to 49,wherein the one or more of the nucleotides is/are modified depending onthe position within the stretch.

EMBODIMENT 51

The nucleic acid molecule of embodiment 50, wherein over the entirelength of the first and/or second stretch or part thereof, a nucleotideat an even position of the stretch is modified.

EMBODIMENT 52

The nucleic acid molecule of any one of embodiments 50 and 51, whereinover the entire length of the first and/or second stretch or partthereof a nucleotide at an uneven position of the stretch is modified.

EMBODIMENT 53

The nucleic acid molecule of any one of embodiments 51 to 52, whereinover the entire length of the first and/or the second stretch or partthereof a nucleotide at an even position of the stretch is modified andwherein over the entire length of the first and/or the second stretch orpart thereof a nucleotide at an even position of the stretch ismodified, wherein the modification of the nucleotide(s) at the evenposition is different from the modification of the nucleotide(s) at theuneven position.

EMBODIMENT 54

The nucleic acid molecule of any one of embodiments 51 to 53, wherein(a) the modification of the nucleotide(s) at the even position is a2′-O-methyl modification and the modification of the nucleotides(s) atthe uneven position is a 2′-F modification, or (b) the modification ofthe nucleotide(s) at the uneven position is a 2′-O-methyl modificationand the modification of the nucleotides(s) at the even position is a2′-F modification.

EMBODIMENT 55

The nucleic acid molecule of any one of embodiments 44 to 50, whereinthe one or more of the nucleotides is/are modified depending on theposition within the strand.

EMBODIMENT 56

The nucleic acid molecule of embodiment 55, wherein over the entirelength of the first and/or second strand or part thereof, a nucleotideat an even position of the strand is modified.

EMBODIMENT 57

The nucleic acid molecule of any one of embodiments 55 and 56, whereinover the entire length of the first and/or second strand or part thereofa nucleotide at an uneven position of the strand is modified.

EMBODIMENT 58

The nucleic acid molecule of any one of embodiments 56 to 57, whereinover the entire length of the first and/or the second strand or partthereof a nucleotide at an even position of the strand is modified andwherein over the entire length of the first and/or the second strand orpart thereof a nucleotide at an even position of the strand is modified,wherein the modification of the nucleotide(s) at the even position isdifferent from the modification of the nucleotide(s) at the unevenposition of the strand.

EMBODIMENT 59

The nucleic acid molecule of any one of embodiments 56 to 58, wherein(a) the modification of the nucleotide(s) at the even position is a2′-O-methyl modification and the modification of the nucleotides(s) atthe uneven position is a 2′-F modification, or (b) the modification ofthe nucleotide(s) at the uneven position is a 2′-O-methyl modificationand the modification of the nucleotides(s) at the even position is a2′-F modification.

EMBODIMENT 60

The nucleic acid molecule of any one of embodiments 41 to 54, whereinthe first stretch comprises at the 5′ end at least one, preferably twonucleotides, wherein the at least one nucleotide is 2′-F modified.

EMBODIMENT 61

The nucleic acid molecule of embodiment 60, wherein after the at leastone nucleotide the immediately following (in 5′->3′ direction)nucleotide and every subsequent second nucleotide is 2′ 0-methlymodified nucleotide over the entire length of the first stretch or partthereof.

EMBODIMENT 62

The nucleic acid molecule of any one of embodiments 60 and 61, whereinstarting after the at least one nucleotide the second following (in5′->3′ direction) nucleotide and every subsequent second nucleotide is2′-F modified nucleotide over the entire length of the first stretch orpart thereof.

EMBODIMENT 63

The nucleic acid molecule of any one of embodiments 41 to 55 and 60 to62, wherein the second stretch comprises at the 5′ end at least one,preferably two nucleotides, wherein the at least one nucleotide is2′-O-methyl modified.

EMBODIMENT 64

The nucleic acid molecule of embodiment 63, wherein after the at leastone nucleotide the immediately following (in 5′->3′ direction)nucleotide and every subsequent second nucleotide is 2′-F modifiednucleotide over the entire length of the second stretch or part thereof.

EMBODIMENT 65

The nucleic acid molecule of any one of embodiments 63 and 64, whereinstarting after the at least one nucleotide the second following (in5′->3′ direction) nucleotide and every subsequent second nucleotide is2′-O-methyl modified nucleotide over the entire length of the firststretch or part thereof.

EMBODIMENT 66

The nucleic acid molecule of any one of embodiments 60 to 65, whereinthe first stretch comprises at the 5′ end at least one, preferably twonucleotides, wherein the at least one nucleotide is 2′-F modified,wherein starting after the at least one nucleotide the second following(in 5′->3′ direction) nucleotide and every subsequent second nucleotideis 2′-F modified nucleotide over the entire length of the first stretchor part thereof, wherein the second stretch comprises at the 5′ end atleast one, preferably two nucleotides, wherein the at least onenucleotide is 2′-O-methyl modified, and wherein starting after the atleast one nucleotide the second following (in 5′->3′ direction)nucleotide and every subsequent second nucleotide is 2′-O-methylmodified nucleotide over the entire length of the first stretch or partthereof.

EMBODIMENT 67

The nucleic acid molecule of any one of embodiments 60 to 66, whereinthe first stretch comprises at its 3′ end two dT nucleotides and thesecond stretch comprises at its 3′ end two dT nucleotides, wherein thetwo dT nucleotides are covalently linked through a phosophorothioatebond.

EMBODIMENT 68

The nucleic acid molecule of any one of embodiments 41 to 49 and 55 to59, wherein the first strand comprises at the 5′ end at least one,preferably two nucleotides, wherein the at least one nucleotide is 2′-Fmodified.

EMBODIMENT 69

The nucleic acid molecule of embodiment 68, wherein after the at leastone nucleotide the immediately following (in 5′->3′ direction)nucleotide and every subsequent second nucleotide is 2′ O-methylmodified nucleotide over the entire length of the first strand or partthereof.

EMBODIMENT 70

The nucleic acid molecule of any one of embodiments 68 and 69, whereinstarting after the at least one nucleotide the second following (in5′->3′ direction) nucleotide and every subsequent second nucleotide is2′-F modified nucleotide over the entire length of the first strand orpart thereof

EMBODIMENT 71

The nucleic acid molecule of any one of embodiments 41 to 49, 55-59 and68 to 70, wherein the second strand comprises at the 5′ end at leastone, preferably two nucleotides, wherein the at least one nucleotide is2′-O-methly modified.

EMBODIMENT 72

The nucleic acid molecule of embodiment 71, wherein after the at leastone nucleotide the immediately following (in 5′->3′ direction)nucleotide and every subsequent second nucleotide is 2′-F modifiednucleotide over the entire length of the second strand or part thereof.

EMBODIMENT 73

The nucleic acid molecule of any one of embodiments 71 and 72, whereinstarting after the at least one nucleotide the second following (in5′->3′ direction) nucleotide and every subsequent second nucleotide is2′-O-methyl modified nucleotide over the entire length of the firststrand or part thereof.

EMBODIMENT 74

The nucleic acid molecule of any one of embodiments 68 to 73, whereinthe first strand comprises at the 5′ end at least one, preferably twonucleotides, wherein the at least one nucleotide is 2′-F modified,wherein starting after the at least one nucleotide the second following(in 5′->3′ direction) nucleotide and every subsequent second nucleotideis 2′-F modified nucleotide over the entire length of the first strandor part thereof, wherein the second strand comprises at the 5′ end atleast one, preferably two nucleotides, wherein the at least onenucleotide is 2′-O-methyl modified, and wherein starting after the atleast one nucleotide the second following (in 5′->3′ direction)nucleotide and every subsequent second nucleotide is 2′-O-methylmodified nucleotide over the entire length of the first strand or partthereof.

EMBODIMENT 75

The nucleic acid molecule of any one of embodiments 68 to 74, whereinthe first strand comprises at its 3′ end two dT nucleotides and thesecond strand comprises at its 3′ end two dT nucleotides, wherein thetwo dT nucleotides are covalently linked through a phosphorothioatebond.

EMBODIMENT 76

The nucleic acid molecule of any one of embodiments 1 to 75, wherein thenucleotide sequence 5′ ACGAGCUGGACCACUGGUC 3′ (SEQ ID NO: 1) is modifiedas follows:

(SEQ ID NO: 6) 5′ acGaGcUgGaCcAcUgGuC 3′,andthe nucleotide sequence 5′ CGAGCUGGACCACUGGU 3′ (SEQ ID NO: 8) ismodified as follows:

(SEQ ID NO: 13) 5′ cgAgCuGgAcCaCuGgU 3′,wherein a minor nucleotide indicates that the nucleotide is 2′-Fmodified and an underlined nucleotide indicates that the nucleotide is2′-O-methyl modified.

EMBODIMENT 77

The nucleic acid molecule of any one of embodiments 1 to 76, wherein thenucleotide sequence 5′ GACCAGUGGUCCAGCUCGU 3′ (SEQ ID NO: 3) is modifiedas follows

(SEQ ID NO: 7) 5′ GAcCaGuGgUcCaGcUcGu 3′,andthe nucleotide sequence 5′ ACCAGUGGUCCAGCUCG 3′ (SEQ ID NO: 9) ismodified as follows

(SEQ ID NO: 14) 5′ ACcAgUgGuCcAgCuCg 3′,wherein a minor nucleotide indicates that the nucleotide is 2′-Fmodified and an underlined nucleotide indicates that the nucleotide is2′-O-methyl modified.

EMBODIMENT 78

The nucleic acid molecule of any one of embodiments 1 to 77, wherein thefirst stretch of contiguous nucleotides comprises the followingnucleotide sequence:

(SEQ ID NO: 6) 5′ acGaGcUgGaCcAcUgGuC 3′ or (SEQ ID NO: 13)5′ cgAgCuGgAcCaCuGgU 3′,wherein a minor nucleotide indicates that the nucleotide is 2′-Fmodified and an underlined nucleotide indicates that the nucleotide is2′-O-methyl modified.

EMBODIMENT 79

The nucleic acid molecule of any one of embodiments 1 to 78, wherein thesecond stretch of contiguous nucleotides comprises the followingnucleotide sequence:

(SEQ ID NO: 7) 5′ GAcCaGuGgUcCaGcUcGu 3′ or (SEQ ID NO: 14)5′ ACcAgUgGuCcAgCuCg 3′,wherein a minor nucleotide indicates that the nucleotide is 2′-Fmodified and an underlined nucleotide indicates that the nucleotide is2′-O-methyl modified.

EMBODIMENT 80

The nucleic acid molecule of any one of embodiments 1 to 79, wherein

a) the first stretch of contiguous nucleotides comprises the followingnucleotide sequence:

(SEQ ID NO: 6) 5′ acGaGcUgGaCcAcUgGuC 3′,andthe second stretch of contiguous nucleotides comprises the followingnucleotide sequence:

(SEQ ID NO: 7) 5′ GAcCaGuGgUcCaGcUcGu 3′,orb) the first stretch of contiguous nucleotides comprises the followingnucleotide sequence:

(SEQ ID NO: 13) 5′ cgAgCuGgAcCaCuGgU 3′,and the second stretch of contiguous nucleotides comprises the followingnucleotide sequence:

(SEQ ID NO: 7) 5′ GAcCaGuGgUcCaGcUcGu 3′,orc) the first stretch of contiguous nucleotides comprises the followingnucleotide sequence:

(SEQ ID NO: 6) 5′ acGaGcUgGaCcAcUgGuC 3′,andthe second stretch of contiguous nucleotides comprises the followingnucleotide sequence:

(SEQ ID NO: 14) 5′ ACcAgUgGuCcAgCuCg 3′,ord) the first stretch of contiguous nucleotides comprises the followingnucleotide sequence:

(SEQ ID NO: 13) 5′ cgAgCuGgAcCaCuGgU 3′,andthe second stretch of contiguous nucleotides comprises the followingnucleotide sequence:

(SEQ ID NO: 14) 5′ ACcAgUgGuCcAgCuCg 3′,wherein a minor nucleotide indicates that the nucleotide is 2′-Fmodified and an underlined nucleotide indicates that the nucleotide is2′-O-methyl modified.

EMBODIMENT 81

The nucleic acid molecule of any one of embodiments 1 to 80, wherein thenucleic acid molecule consists of a

a) a first stretch of contiguous nucleotides comprises the followingnucleotide sequence:

(SEQ ID NO: 6) 5′ acGaGcUgGaCcAcUgGuC 3′,anda second stretch of contiguous nucleotides comprises the followingnucleotide sequence:

(SEQ ID NO: 7) 5′ GAcCaGuGgUcCaGcUcGu 3′,orb) a first stretch of contiguous nucleotides comprises the followingnucleotide sequence:

(SEQ ID NO: 13) 5′ cgAgCuGgAcCaCuGgU 3′,anda second stretch of contiguous nucleotides comprises the followingnucleotide sequence:

(SEQ ID NO: 7) 5′ GAcCaGuGgUcCaGcUcGu 3′,orc) a first stretch of contiguous nucleotides comprises the followingnucleotide sequence:

(SEQ ID NO: 6) 5′ acGaGcUgGaCcAcUgGuC 3′,anda second stretch of contiguous nucleotides comprises the followingnucleotide sequence:

(SEQ ID NO: 14) 5′ ACcAgUgGuCcAgCuCg 3′,ord) a first stretch of contiguous nucleotides comprises the followingnucleotide sequence:

(SEQ IO NO: 13) 5′ cgAgCuGgAcCaCuGgU 3′,anda second stretch of contiguous nucleotides comprises the followingnucleotide sequence:

(SEQ ID NO: 14) 5′ ACcAgUgGuCcAgCuCg 3′,wherein a minor nucleotide indicates that the nucleotide is 2′-Fmodified and an underlined nucleotide indicates that the nucleotide is2′-O-methyl modified.

EMBODIMENT 82

The nucleic acid molecule of any one of embodiments 1 to 81, wherein thenucleic acid molecule consists of

a) a first stretch of contiguous nucleotides comprises the followingnucleotide sequence:

(SEQ ID NO: 6) 5′ acGaGcUgGaCcAcUgGuC 3′,anda second stretch of contiguous nucleotides comprises the followingnucleotide sequence:

(SEQ ID NO: 7) 5′ GAcCaGuGgUcCaGcUcGu 3′,orb) a first stretch of contiguous nucleotides comprises the followingnucleotide sequence:

(SEQ IO NO: 13) 5′ cgAgCuGgAcCaCuGgU 3′,anda second stretch of contiguous nucleotides comprises the followingnucleotide sequence:

(SEQ ID NO: 7) 5′ GAcCaGuGgUcCaGcUcGu 3′,orc) a first stretch of contiguous nucleotides comprises the followingnucleotide sequence:

(SEQ ID NO: 6) 5′ acGaGcUgGaCcAcUgGuC 3′,anda second stretch of contiguous nucleotides comprises the followingnucleotide sequence:

(SEQ ID NO: 14) 5′ ACcAgUgGuCcAgCuCg 3′,ord) a first stretch of contiguous nucleotides comprises the followingnucleotide sequence:

(SEQ IO NO: 13) 5′ cgAgCuGgAcCaCuGgU 3′,anda second stretch of contiguous nucleotides comprises the followingnucleotide sequence:

(SEQ ID NO: 14) 5′ ACcAgUgGuCcAgCuCg 3′,wherein a minor nucleotide indicates that the nucleotide is 2′-Fmodified and an underlined nucleotide indicates that the nucleotide is2′-O-methyl modified.

EMBODIMENT 83

The nucleic acid molecule of any one of embodiments 1 to 81, wherein thenucleic acid molecule consists of a

a) a first stretch of contiguous nucleotides comprises the followingnucleotide sequence:

(SEQ ID NO: 69) 5′ acGaGcUgGaCcAcUgGuCdTsdT 3′,anda second stretch of contiguous nucleotides comprises the followingnucleotide sequence:

(SEQ ID NO: 70) 5′ GAcCaGuGgUcCaGcUcGudTsdT 3′,orb) a first stretch of contiguous nucleotides comprises the followingnucleotide sequence:

(SEQ IO NO: 71) 5′ cgAgCuGgAcCaCuGgUdTsdT 3′,anda second stretch of contiguous nucleotides comprises the followingnucleotide sequence:

(SEQ ID NO: 70) 5′ GAcCaGuGgUcCaGcUcGudTsdT 3′,orc) a first stretch of contiguous nucleotides comprises the followingnucleotide sequence:

(SEQ ID NO: 69) 5′ acGaGcUgGaCcAcUgGuCdTsdT 3′,anda second stretch of contiguous nucleotides comprises the followingnucleotide sequence:

(SEQ ID NO: 72) 5′ ACcAgUgGuCcAgCuCgdTsdT 3′,ord) a first stretch of contiguous nucleotides comprises the followingnucleotide sequence:

(SEQ IO NO: 71) 5′ cgAgCuGgAcCaCuGgUdTsdT 3′,anda second stretch of contiguous nucleotides comprises the followingnucleotide sequence:

(SEQ ID NO: 72) 5′ ACcAgUgGuCcAgCuCgdTsdT 3′,wherein a minor nucleotide indicates that the nucleotide is 2′-Fmodified and an underlined nucleotide indicates that the nucleotide is2′-O-methyl modified and wherein dTsdT indicates that at the 3′ end adinucleotide is attached consisting of two dTs, wherein said two dTs arecovalently linked through a phosphorothioate bond.

EMBODIMENT 84

The nucleic acid molecule of any one of embodiments 1 to 83, for use ina method for the treatment and/or prevention of a disease.

EMBODIMENT 85

The nucleic acid molecule of embodiment 84, wherein the disease is adisease which can be treated by decreasing the expression of the Notch 1gene and more specifically by decreasing the translation of the mRNAcoding for Notch 1.

EMBODIMENT 86

The nucleic acid molecule of any one of embodiments 84 to 85, whereinthe disease is selected from the group comprising esophageal cancer,oral squamous cell carcinoma, head and neck cancer, tongue cancer,leukemia, renal cell carcinoma, gastric cancer, colon adenocarcinoma,endometrial cancer/uterine corpus, cervical cancer/uterine cervix,intrahepatic cholangiocarcinoma, hepatocellular carcinoma, osteosarcoma,urinary bladder carcinoma, malignant melanoma, thyroid cancer, lungadenocarcinoma, prostate cancer, breast cancer, ovarian cancer,pancreatic cancer and glioma.

EMBODIMENT 87

The nucleic acid molecule of any one of embodiments 84 to 86, whereinthe method comprises further the administration of a pharmaceuticallyactive agent.

EMBODIMENT 88

The nucleic acid molecule of embodiment 87, wherein the pharmaceuticallyactive agent is a cytostatic.

EMBODIMENT 89

The nucleic acid molecule of embodiment 88, wherein the pharmaceuticallyactive agent is selected from the group comprising gemcitabine,docetaxel, cisplaint, oxaliplatin, 5-fluorouracil, irinotecan,paclitaxel, dexamethasone and temozolomide.

EMBODIMENT 90

The nucleic acid molecule of any one of embodiments 1 to 83, for use ina method for restoring drug sensitivity of cancer cells.

EMBODIMENT 91

The nucleic acid molecule of embodiment 90, wherein drug sensitivity isdrug sensitivity mediated and/or involving NF-kappaB cascade.

EMBODIMENT 92

Use of a nucleic acid molecule of any one of embodiments 1 to 83, forthe manufacture of a medicament for the treatment and/or prevention of adisease.

EMBODIMENT 93

Use of a embodiment 92, wherein the disease is a disease which can betreated by decreasing the expression of the Notch 1 gene and morespecifically by decreasing the translation of the mRNA coding for Notch1.

EMBODIMENT 94

Use of any one of embodiments 92 to 93, wherein the disease is selectedfrom the group comprising esophageal cancer, oral squamous cellcarcinoma, head and neck cancer, tongue cancer, leukemia, renal cellcarcinoma, gastric cancer, colon adenocarcinoma, endometrialcancer/uterine corpus, cervical cancer/uterine cervix, intrahepaticcholangiocarcinoma, hepatocellular carcinoma, osteosarcoma, urinarybladder carcinoma, malignant melanoma, thyroid cancer, lungadenocarcinoma, prostate cancer, breast cancer, ovarian cancer,pancreatic cancer and glioma.

EMBODIMENT 95

Use of any one of embodiments 92 to 94, wherein the medicament is foradministration together with a further pharmaceutically active agent.

EMBODIMENT 96

Use of embodiment 95, wherein the pharmaceutically active agent is acytostatic.

EMBODIMENT 97

Use of embodiment 96, wherein the pharmaceutically active agent is

selected from the group comprising gemcitabine, docetaxel, cisplaint,oxaliplatin, 5-fluorouracil, irinotecan, paclitaxel, dexamethasone andtemozolomide.

EMBODIMENT 98

Use of a nucleic acid molecule of any one of embodiments 1 to 83, in themanufacture of an agent for restoring drug sensitivity of cancer cells.

EMBODIMENT 99

Use of embodiment 98, wherein drug sensitivity is drug sensitivitymediated and/or involving NF-kappaB cascade.

EMBODIMENT 100

A nanoemulsion comprising a discontinuous phase and a continuous aqueousphase and a nucleic acid molecule according to any one of embodiments 1to 83.

EMBODIMENT 101

The nanoemulsion of embodiment 100, wherein the discontinuous phasecomprises a perfluorocarbon phase.

EMBODIMENT 102

The nanoemulsion of any one of embodiments 1 to 101, wherein thenanoemulsion comprises an endocytosis enhancing surface, preferably theendocytosis enhancing surface comprises an endocytosis enhancingcomponent, wherein the endocytosis enhancing component is selected fromthe group comprising at least one compound inducing cellular uptake ofthe nanoemulsion or particles of the nanoemulsion via endocytosis.

EMBODIMENT 103

The nanoemulsion of any one of embodiments 100 to 102, for use in thetreatment and/or prevention of a disease.

EMBODIMENT 104

The nanoemulsion of any one of embodiments 100 to 102, for use in amethod for restoring drug sensitivity of cancer cells.

EMBODIMENT 105

Use of a nanoemulsion of any one of embodiments 100 to 102, for themanufacture of a medicament for the treatment and/or prevention of adisease.

EMBODIMENT 106

Use of a nanoemulsion of any one of embodiments 100 to 102, in themanufacture of an agent for restoring drug sensitivity of cancer cells.

EMBODIMENT 107

A pharmaceutical composition comprising a nucleic acid molecule of anyone of embodiments 1 to 83 and/or a nanoemulsion of any one ofembodiments 100 to 102, and a pharmaceutically acceptable excipient.

EMBODIMENT 108

The pharmaceutical composition of embodiment 107, for use in thetreatment and/or prevention of a disease.

EMBODIMENT 109

The pharmaceutical composition of embodiment 107, for use in a methodfor restoring drug sensitivity of cancer cells.

EMBODIMENT 110

A method for the treatment and/or prevention of a disease, wherein themethod comprises the administration to a subject of a nucleic acid ofany one of embodiments 1 to 83, a nanoemulsion of any one of embodiments100 to 102, and/or a pharmaceutical composition of embodiment 107.

EMBODIMENT 111

A method for restoring drug sensitivity of cancer cells, wherein themethod comprises the administration to a subject of a nucleic acid ofany one of embodiments 1 to 83, a nanoemulsion of any one of embodiments100 to 102, and/or a pharmaceutical composition of embodiment 107,wherein the subject is suffering from cancer and cells of the cancer aredrug-resistant.

The present inventors have surprisingly found that a nucleic acidmolecule comprising a double-stranded structure,

wherein the double-stranded structure is formed by a first strand and asecond strand,wherein the first strand comprises a first stretch of contiguousnucleotides and the second strand comprises a second stretch ofcontiguous nucleotides,wherein the first stretch of contiguous nucleotides comprisesa) a nucleotide sequence, wherein the nucleotide sequence is at least63% identical to

(i) nucleotide sequence (SEQ ID NO: 1) 5′ ACGAGCUGGACCACUGGUC 3′ or (ii)nucleotide sequence (SEQ ID NO: 8) 5′ CGAGCUGGACCACUGGU 3′;orb) a nucleotide sequence, wherein the nucleotide sequence comprises atleast a stretch of 8 or 9 nucleotides of

(i) nucleotide sequence (SEQ ID NO: 1) 5′ ACGAGCUGGACCACUGGUC 3′ or (ii)nucleotide sequence (SEQ ID NO: 8) 5′ CGAGCUGGACCACUGGU 3′is capable of causing post-transcriptional silencing of a gene and RNAinterference in particular. This nucleic acid, including all of itsembodiments, will be referred to herein as the nucleic acid molecule ofthe invention.

It is within the invention that the nucleic acid molecule of theinvention is a small interfering RNA (siRNA). Such siRNA is aparticularly preferred embodiment of the nucleic acid molecule of theinvention. In an embodiment the siRNA is directed to an expressed RNAtranscript of Notch 1 (sometimes referred to as a “target nucleic acid”herein). As preferably used herein, the terms “silence” and “knock-down”when referring to gene expression means a reduction in gene expression.The present invention further relates to processes for making thenucleic acid molecule of the invention.

In an embodiment of the invention, the target nucleic acid is an RNAexpressed from a mammalian Notch 1 gene. In one embodiment, the targetnucleic acid is an RNA expressed from mouse Notch 1. In anotherembodiment, the target nucleic acid is an RNA expressed from humanNotch 1. In another embodiment, the target nucleic acid is a human Notch1 mRNA. In another embodiment, the target nucleic acid is a human Notch1 hnRNA. In another embodiment, the target nucleic acid is an mRNAcomprising the sequence of SEQ ID NO: 12.

In an embodiment of the present invention the nucleic acid molecule isnot forming a double-stranded structure. In such embodiment the nucleicacid molecule is either formed by two separate single strands which maybe present individually, i.e. in a non-hybridized state so that thedouble-stranded structure is not formed, or in a hybridized form where adouble-stranded structure is formed which is different from thedouble-stranded structure which is required so as to mediate or triggerRNA interference. Alternatively, the nucleic acid molecule forming thedouble-stranded structure is a single strand nucleic acid molecule,wherein the nucleic acid molecule is not folding back on itself suchthat the double-stranded structure is formed or such that adouble-stranded structure is formed which is different from thedouble-stranded structure which is required so as to mediate or triggerRNA interference. In a more preferred embodiment the double-strandedstructure is formed under in vivo conditions, and more specifically uponadministration of the nucleic acid molecule to a subject, preferably amammal or mammalian cell.

The siRNA of the present invention are suitable to inhibit theexpression of Notch 1. The siRNA according to the present invention is,thus, suitable to trigger the RNA interference response resulting in thereduction of the Notch 1 mRNA in a mammalian cell. The siRNA accordingto the present invention are further suitable to decrease the expressionof Notch 1 protein by decreasing gene expression at the level of mRNA.

siRNA Design:

An siRNA of the present invention comprises two strands of a nucleicacid, a first strand, which is also referred to as antisense strand,comprising a first stretch of contiguous nucleotides, which is alsoreferred to as antisense stretch, and a second strand, which is alsoreferred to as sense strand, comprising a second stretch of contiguousnucleotides which is also referred to as sense stretch. The nucleic acidnormally consists of ribonucleotides or modified ribonucleotideshowever; the nucleic acid may comprise deoxynucleotides (DNA) asdescribed herein. The siRNA further comprises a double-stranded nucleicacid portion or duplex region formed by all or a portion of theantisense strand or the antisense stretch and all or a portion of thesense strand or the sense stretch. Such double-stranded nucleic acidportion or duplex region is herein also referred to as double-strandedstructure. The portion of the antisense strand or of the antisensestretch forming the duplex region with the sense strand or with theantisense stretch is the antisense strand duplex region or the antisensestretch region or simply, the antisense duplex region, and the portionof the sense strand or of the sense stretch forming the duplex regionwith the antisense strand or the antisense stretch is the sense strandduplex region is the sense stretch duplex region or simply, the senseduplex region. The duplex region is defined as beginning with the firstbase pair formed between the antisense strand or the antisense stretchand the sense strand of the sense stretch and ending with the last basepair formed between the antisense strand or the antisense stretch andthe sense strand or the sense stretch, inclusive. The portion of thesiRNA on either side of the duplex region is the flanking regions. Theportion of the antisense strand or of the antisense stretch on eitherside of the antisense duplex region is the antisense flanking regions.The portion of the antisense strand or antisense stretch 5′ to theantisense duplex region is the antisense 5′ flanking region. The portionof the antisense strand or antisense stretch 5′ to the antisense duplexregion is the antisense 3′ flanking region. The portion of the sensestrand or of the sense stretch on either side of the sense duplex regionis the sense flanking regions. The portion of the sense strand or of thesense stretch 5′ to the sense duplex region is the sense 5′ flankingregion. The portion of the sense strand 5′ or of the sense stretch tothe sense duplex region is the sense 3′ flanking region.

Identity:

In an embodiment, identity of one nucleotide sequence to anothernucleotide sequence is an indication of how many nucleotides are sharedbetween both the one nucleotide sequence and the another nucleotidesequence. Identity is expresses as the ratio of the number ofnucleotides of the one sequence shared with the another nucleotidesequence to the total number of nucleotides of the another nucleotidesequence. The maximum value of identity is 100%. It will be acknowledgedby a person skilled in the art that depending on the length of the onenucleotide sequence and of the another nucleotide sequence on the onehand and the number of nucleotides shared between the one nucleotidesequence and the another nucleotide sequence identity is not always aninteger. If such calculated ratio is not an integer, the identity isnevertheless preferably indicated as the integer which gets as close aspossible to the calculated ratio and which makes technically sense.According to the present invention, the identity may be 63%, 64%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or 100%.

Complementarity:

According to the invention, the antisense duplex region and the senseduplex region may be fully complementary and are at least partiallycomplementary to each other. Such complementarity is based onWatson-Crick base pairing (i.e., A:U and G:C base pairing). Depending onthe length of a nucleic acid molecule of the invention and an siRNA inparticular a perfect match in terms of base complementarity between theantisense and sense duplex regions is not necessarily required however,the antisense and sense strands must be able to hybridize underphysiological conditions.

In one embodiment, the complementarity between the antisense strand andsense strand is perfect, i.e. no nucleotide mismatches oradditional/deleted nucleotides in either strand.

In one embodiment, the complementarity between the antisense stretch andsense stretch is perfect, i.e. no nucleotide mismatches oradditional/deleted nucleotides in either stretch.

In one embodiment, the complementarity between the antisense duplexregion and sense duplex region is perfect, i.e. no nucleotide mismatchesor additional/deleted nucleotides in the duplex region of either strand.

In another embodiment, the complementarity between the antisense duplexregion and the sense duplex region is not perfect. In one embodiment,the identity between the antisense duplex region and the complementarysequence of the sense duplex region is selected from the groupconsisting of at least 75%, 80%, 85%, 90% and 95%; wherein a siRNAcomprising the antisense duplex region and the sense duplex region issuitable for reducing expression of Notch 1. In another embodiment, thesiRNA, wherein the identity between the antisense duplex region andcomplementary sequence of the sense duplex region is selected from thegroup consisting of at least 75%, 80%, 85%, 90% and 95%, is able toreduce expression of Notch 1 by at least 25%, 50% or 75% of acomparative siRNA having a duplex region with perfect identity betweenthe antisense duplex region and the sense duplex region. As used hereinthe term “comparative siRNA” is a siRNA that is identical to the siRNAto which it is being compared, except for the specified difference, andwhich is tested under identical conditions.

RNAi using siRNA involves the formation of a duplex region between allor a portion of the antisense strand or antisense stretch and a portionof the target nucleic acid. The portion of the target nucleic acid thatforms a duplex region with the antisense strand or antisense stretch,defined as beginning with the first base pair formed between theantisense strand or antisense stretch and the target sequence and endingwith the last base pair formed between the antisense strand or antisensestretch and the target sequence, inclusive, is the target nucleic acidsequence or simply, target sequence. The duplex region formed betweenthe antisense strand or antisense stretch and the sense strand or sensestretch may, but need not be the same as the duplex region formedbetween the antisense strand or antisense stretch and the targetsequence. That is, the sense strand or sense stretch may have a sequencedifferent from the target sequence however; the antisense strand orantisense stretch must be able to form a duplex structure with both thesense strand or sense stretch and the target sequence.

In one embodiment, the complementarity between the antisense strand orantisense stretch and the target sequence is perfect, i.e. no nucleotidemismatches or additional/deleted nucleotides in either nucleic acid.

In one embodiment, the complementarity between the antisense duplexregion, i.e. the portion of the antisense strand or antisense stretchforming a duplex region with the sense strand or sense stretch, and thetarget sequence is perfect, i.e. no nucleotide mismatches oradditional/deleted nucleotides in either nucleic acid.

In another embodiment, the complementarity between the antisense duplexregion and the target sequence is not perfect. In one embodiment, theidentity between the antisense duplex region and the complementarysequence of the target sequence is selected from the group consisting ofat least 75%, 80%, 85%, 90% or 95%, wherein a siRNA comprising theantisense duplex region is suitable for reducing expression of Notch 1.In another embodiment, the siRNA, wherein the identity between theantisense duplex region and complementary sequence of the targetsequence is selected from the group consisting of at least 75%, 80%,85%, 90% and 95%, is able to reduce expression of Notch 1 by at least25%, 50% or 75% of a comparative siRNA with perfect identity to theantisense strand or the antisense stretch and target sequence.

In another embodiment, the siRNA of the invention comprises a duplexregion wherein the antisense duplex region has a number of nucleotidesselected from the group consisting of 1, 2, 3, 4 and 5 that are notbase-paired to a nucleotide in the sense duplex region, and wherein saidsiRNA is suitable for reducing expression of Notch 1. Lack ofbase-pairing is due to either lack of complementarity between bases,i.e., no Watson-Crick base pairing, or because there is no correspondingnucleotide on either the antisense duplex region or the sense duplexregion such that a bulge is created. In one embodiment, a siRNAcomprising an antisense duplex region having a number of nucleotidesselected from the group consisting of 1, 2, 3, 4 and 5 that are notbase-paired to the sense duplex region, is able to reduce expression ofNotch 1 by at least 25%, 50%, 75% of a comparative siRNA wherein allnucleotides of said antisense duplex region are base paired with allnucleotides of said sense duplex region.

In another embodiment, the antisense strand or the antisense stretch hasa number of nucleotides selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10that do not base-pair to the sense strand or the sense stretch, andwherein a siRNA comprising said antisense strand is suitable forreducing expression of Notch 1. Lack of complementarity is due to eitherlack of complementarity between bases or because there is nocorresponding nucleotide on either the antisense strand, or theantisense stretch, or the sense strand, or the sense stretch. The lackof a corresponding nucleotide results in either a single-strandedoverhang or a bulge (if in the duplex region), in either the antisensestrand, or the antisense stretch, or the sense strand, or the sensestretch. In one embodiment, a siRNA comprising an antisense strand or anantisense stretch having a number of nucleotides selected from 1, 2, 3,4, 5, 6, 7, 8, 9 or 10 that do not base pair to the sense strand or thesense stretch, is able to reduce expression of Notch 1 by at least 25%,50%, 75% of a comparative siRNA wherein all nucleotides of saidantisense strand or said antisense stretch are complementary to allnucleotides of the sense strand of the sense stretch. In one embodiment,a siRNA comprising an antisense strand or an antisense stretch having anumber of nucleotides selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 thatare mismatched to the target sequence, is able to reduce expression ofNotch 1 by at least 25%, 50%, 75% of a comparative siRNA wherein allnucleotides of said antisense strand or said antisense strand arecomplementary to all nucleotides of said sense strand or said sensestretch. In another embodiment, all of the mismatched nucleotides areoutside the duplex region.

In another embodiment, the antisense duplex region has a number ofnucleotides selected from 1, 2, 3, 4 or 5 that do not base-pair to thesense duplex region, and wherein a siRNA comprising said antisenseduplex region is suitable for reducing expression of Notch 1. Lack ofcomplementarity is due to either lack of complementarity between basesor because there is no corresponding nucleotide on either the antisenseduplex region or the sense duplex region such that a bulge in created ineither the antisense duplex region or the sense duplex region. In oneembodiment, a siRNA comprising an antisense duplex region having anumber of nucleotides selected from the group consisting of 1, 2, 3, 4and 5 that do not base pair to the sense duplex region, is able toreduce expression of Notch 1 by at least 25%, 50%, 75% of a comparativesiRNA wherein all nucleotides of said antisense duplex region arecomplementary to all of the nucleotides of said sense duplex region.

In another embodiment, the antisense strand has a number of nucleotidesselected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 that do not base-pair tothe target sequence, and wherein a siRNA comprising said antisensestrand is suitable for reducing expression of Notch 1. Lack ofcomplementarity is due to either lack of complementarity between basesor because there is no corresponding nucleotide on either the antisensestrand, or the antisense stretch, or the target sequence. The lack of acorresponding nucleotide results in a bulge in either the antisensestrand, or the antisense stretch, or the target sequence. In oneembodiment, a siRNA comprising an antisense strand or an antisensestretch having a number of nucleotides selected from 1, 2, 3, 4, 5, 6,7, 8, 9 or 10 do not base pair to the target sequence, is able to reduceexpression of Notch 1 by at least 25%, 50%, 75% of a comparative siRNAwherein all nucleotides of said antisense strand or antisense stretchare complementary to all nucleotides of said target sequence. In oneembodiment, a siRNA comprising an antisense strand or an antisensestretch having a number of nucleotides selected from 1, 2, 3, 4, 5, 6,7, 8, 9 or 10 that are mismatched to the target sequence, is able toreduce expression of Notch 1 by at least 25%, 50% or 75% of acomparative siRNA wherein all nucleotides of said antisense strand or ofsaid antisense stretch are complementary to all nucleotides of saidtarget sequence.

In another embodiment, the complementarity between an antisense duplexregion and both a sense duplex region and a target sequence of an siRNAis such that the antisense duplex region and the sense duplex region orthe target sequence hybridize to one another under physiologicalconditions (37° C. in a physiological buffer) and the siRNA is suitablefor reducing expression of Notch 1. In one embodiment, the siRNAcomprising an antisense duplex region that hybridizes to a sense duplexregion and a target sequence under physiological conditions, is able toreduce expression of Notch 1 by at least 25%, 50%, 75% of a comparativesiRNA with perfect complementarity between the antisense strand or theantisense stretch and target sequence.

In another aspect, the complementarity between an antisense duplexregion and a sense duplex region of a siRNA is such that the antisenseduplex region and sense duplex region hybridize under the followingconditions: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 70° C., and issuitable for reducing expression of Notch 1. In one embodiment, thesiRNA comprising an antisense duplex region and a sense duplex regionthat hybridize to one another under the conditions 400 mM NaCl, 40 mMPIPES pH 6.4, 1 mM EDTA, 70° C., is able to reduce expression of Notch 1by at least 25%, 50%, 75% of a comparative siRNA with perfectcomplementarity between the antisense duplex region and sense duplexregion.

In another embodiment, the complementarity between an antisense strandor an antisense stretch of a siRNA and a target sequence is such thatthe antisense strand or antisense stretch and target sequence hybridizeunder the following conditions: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mMEDTA, 70° C. and wherein the siRNA is suitable for reducing expressionof Notch 1. In one embodiment, the siRNA comprising an antisense strandor an antisense stretch that hybridizes to the target sequence under thefollowing conditions: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 70°C., is able to reduce expression of Notch 1 by at least 25%, 50%, 75% ofa comparative siRNA with perfect complementarity between the antisensestrand or the antisense stretch and the target sequence.

Length:

RNA interference is observed using long nucleic acid moleculescomprising several dozen or hundreds of base pairs, although shorterRNAi molecules are generally preferred.

In one embodiment, the length of the siRNA duplex region is selectedfrom the group consisting of about 16 to 35, 16 to 30, 17 to 35, 17 to30, 17 to 25, 17 to 24, 18 to 29, 18 to 25, 18 to 24, 18 to 23, 19 to25, 19 to 24, 19 to 23, 20 to 25, 20 to 24, 21 to 25 and 21 to 24 basepairs. In one embodiment, the length of the siRNA duplex region isselected from the group consisting of about 16 to 35, 16 to 30, 17 to35, 17 to 30, 17 to 25, 17 to 24, 18 to 29, 18 to 25, 18 to 24, 18 to23, 19 to 25, 19 to 24, 19 to 23, 20 to 25, 20 to 24, 21 to 25 and 21 to24 consecutive base pairs. In another embodiment, the length of thesiRNA duplex region is selected from the group consisting of 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 and 35base pairs. In another embodiment, the length of the siRNA duplex regionis selected from the group consisting of 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 and 35 consecutive basepairs.

In one embodiment, the length of the antisense strand is selected fromthe group consisting of about 13 to 35, 16 to 35, 16 to 30, 17 to 35, 17to 30, 17 to 25, 17 to 24, 18 to 29, 18 to 25, 18 to 24, 18 to 23, 19 to25, 19 to 24, 19 to 23, 20 to 25, 20 to 24, 21 to 25 and 21 to 24nucleotides. In one embodiment, the length of the antisense stand isselected from the group consisting of 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 and 35 nucleotides.

In one embodiment, the length of the antisense stretch is selected fromthe group consisting of about 13 to 35, 16 to 35, 16 to 30, 17 to 35, 17to 30, 17 to 25, 17 to 24, 18 to 29, 18 to 25, 18 to 24, 18 to 23, 19 to25, 19 to 24, 19 to 23, 20 to 25, 20 to 24, 21 to 25 and 21 to 24nucleotides. In one embodiment, the length of the antisense stretch isselected from the group consisting of 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 and 35nucleotides.

In one embodiment, the length of the sense strand is selected from thegroup consisting of about 13 to 35, 16 to 35, 16 to 30, 17 to 35, 17 to30, 17 to 25, 17 to 24, 18 to 29, 18 to 25, 18 to 24, 18 to 23, 19 to25, 19 to 24, 19 to 23, 20 to 25, 20 to 24, 21 to 25 and 21 to 24nucleotides. In one embodiment, the length of the sense stand isselected from the group consisting of 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 and 35nucleotides.

In one embodiment, the length of the sense stretch is selected from thegroup consisting of about 13 to 35, 16 to 35, 16 to 30, 17 to 35, 17 to30, 17 to 25, 17 to 24, 18 to 29, 18 to 25, 18 to 24, 18 to 23, 19 to25, 19 to 24, 19 to 23, 20 to 25, 20 to 24, 21 to 25 and 21 to 24nucleotides. In one embodiment, the length of the sense stretch isselected from the group consisting of 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 and 35nucleotides.

In one embodiment, the length of the antisense strand and the length ofthe sense strand are independently selected from the group consisting ofabout 13 to 35, 16 to 35, 16 to 30, 17 to 35, 17 to 30, 17 to 25, 17 to24, 18 to 29, 18 to 25, 18 to 24, 18 to 23, 19 to 25, 19 to 24, 19 to23, 20 to 25, 20 to 24, 21 to 25 and 21 to 24 nucleotides. In oneembodiment, the length of the antisense strand and the length of thesense stand are independently selected from the group consisting of 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34 and 35 nucleotides. In one embodiment, the antisense strandand the sense strand are equal in length. In another embodiment, theantisense strand and the sense stand are equal in length, wherein thelength is selected from the group consisting of 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 and 35nucleotides.

In one embodiment, the length of the antisense stretch and the length ofthe sense stretch are independently selected from the group consistingof about 13 to 35, 16 to 35, 16 to 30, 17 to 35, 17 to 30, 17 to 25, 17to 24, 18 to 29, 18 to 25, 18 to 24, 18 to 23, 19 to 25, 19 to 24, 19 to23, 20 to 25, 20 to 24, 21 to 25 and 21 to 24 nucleotides. In oneembodiment, the length of the antisense stretch and the length of thesense stretch are independently selected from the group consisting of13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34 and 35 nucleotides. In one embodiment, the antisensestretch and the sense stretch are equal in length. In anotherembodiment, the antisense stretch and the sense stretch are equal inlength, wherein the length is selected from the group consisting of 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34 and 35 nucleotides.

In one embodiment, the length of the antisense strand or the antisensestretch is selected from the group consisting of about 17 to 35, 17 to30, 17 to 25, 17 to 24, 18 to 29, 18 to 25, 18 to 24, 18 to 23, 19 to25, 19 to 24, 19 to 23, 20 to 25, 20 to 24, 21 to 25 and 21 to 24nucleotides, wherein the antisense strand or antisense stretch comprisesthe nucleotide sequence of SEQ ID NO: 8 or 1.

In one embodiment, the length of the antisense strand or the antisensestretch is selected from the group consisting of about 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 and 35nucleotides, wherein the antisense strand or the antisense stretchcomprises the nucleotide sequence of SEQ ID NOs: 8 or 1.

In one embodiment, the length of the sense strand or the sense stretchis selected from the group consisting of about 13 to 35, 16 to 35, 16 to30, 17 to 35, 17 to 30, 17 to 25, 17 to 24, 18 to 29, 18 to 25, 18 to24, 18 to 23, 19 to 25, 19 to 24, 19 to 23, 20 to 25, 20 to 24, 21 to 25and 21 to 24 nucleotides, wherein the sense strand or the sense stretchcomprises the nucleotide sequence of SEQ ID NOs: 9 or 3.

In one embodiment, the length of the sense strand or of the sensestretch is selected from the group consisting of about 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 and 35nucleotides, wherein the sense strand comprises the nucleotide sequenceof SEQ ID NOs: 9 or 3.

In one embodiment, the length of the antisense strand or of theantisense stretch and the length of the sense strand or the sensestretch are independently selected from the group consisting of about 13to 35, 16 to 35, 16 to 30, 17 to 35, 17 to 30, 17 to 25, 17 to 24, 18 to29, 18 to 25, 18 to 24, 18 to 23, 19 to 25, 19 to 24, 19 to 23, 20 to25, 20 to 24, 21 to 25 and 21 to 24 nucleotides, wherein the antisensestrand or antisense stretch comprises the nucleotide sequence of SEQ IDNO. NOs: 8 or 1, and wherein the sense strand or the sense stretchcomprises the nucleotide sequence of SEQ ID NOs: 9 or 3.

In one embodiment, the length of the antisense strand and the length ofthe sense stand are independently selected from the group consisting of17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34and 35 nucleotides, wherein the antisense strand comprises thenucleotide sequence of SEQ ID NO. NOs: 8 or 1, and wherein the sensestrand comprises the nucleotide sequence of SEQ ID NOs: 9 or 3.

In one embodiment, the length of the antisense stretch and the length ofthe sense stretch and are independently selected from the groupconsisting of 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34 and 35 nucleotides, wherein the antisense stretchcomprises the nucleotide sequence of SEQ ID NO. NOs: 8 or 1, and whereinthe sense stretch comprises the nucleotide sequence of SEQ ID NOs: 9 or3.

In one embodiment, the antisense strand and the sense strand are equalin length, wherein the antisense strand comprises the nucleotidesequence of SEQ ID NO. NOs: 8 or 1, and wherein the sense strandcomprises the nucleotide sequence of SEQ ID NOs: 9 or 3.

In one embodiment, the antisense stretch and the sense stretch are equalin length, wherein the antisense stretch comprises the nucleotidesequence of SEQ ID NO. NOs: 8 or 1, and wherein the sense stretchcomprises the nucleotide sequence of SEQ ID NOs: 9 or 3.

In another embodiment, the antisense strand and the sense stand areequal in length, wherein the length is selected from the groupconsisting of 17 to 35, 17 to 30, 17 to 25, 17 to 24, 18 to 29, 18 to25, 18 to 24, 18 to 23, 19 to 25, 19 to 24, 19 to 23, 20 to 25, 20 to24, 21 to 25 and 21 to 24 nucleotides, wherein the antisense strandcomprises the nucleotide sequence of SEQ ID NO. NOs: 8 or 1, and whereinthe sense strand comprises the nucleotide sequence of SEQ ID NOs: 9 or3.

In another embodiment, the antisense stretch and the sense stretch areequal in length, wherein the length is selected from the groupconsisting of 17 to 35, 17 to 30, 17 to 25, 17 to 24, 18 to 29, 18 to25, 18 to 24, 18 to 23, 19 to 25, 19 to 24, 19 to 23, 20 to 25, 20 to24, 21 to 25 and 21 to 24 nucleotides, wherein the antisense stretchcomprises the nucleotide sequence of SEQ ID NO. NOs: 8 or 1, and whereinthe sense stretch comprises the nucleotide sequence of SEQ ID NOs: 9 or3.

In another embodiment, the antisense strand and the sense stand areequal in length, wherein the length is selected from the groupconsisting of 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34 and 35 nucleotides, wherein the antisense strandcomprises the nucleotide sequence of SEQ ID NO. NOs: 8 or 1, and whereinthe sense strand comprises the nucleotide sequence of SEQ ID NOs: 9 or3.

In another embodiment, the antisense stretch and the sense stretch areequal in length, wherein the length is selected from the groupconsisting of 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34 and 35 nucleotides, wherein the antisense stretchcomprises the nucleotide sequence of SEQ ID NO. NOs: 8 or 1, and whereinthe sense stretch comprises the nucleotide sequence of SEQ ID NOs: 9 or3.

Certain embodiments provide for antisense and sense strand combinations(identified by SEQ ID NO:): 8 and 9; 1 and 3; 4 and 5; 6 and 7; and 10and 11.

Certain embodiments provide for antisense and sense stretch combinations(identified by SEQ ID NO:): 8 and 9; 1 and 3; 4 and 5; 6 and 7; and 10and 11.

Ends (Overhangs and Blunt Ends):

The siRNA of the present invention may comprise an overhang or be bluntended. An “overhang” as used herein has its normal and customary meaningin the art, i.e., a single stranded portion of a nucleic acid thatextends beyond the terminal nucleotide of a complementary strand orstretch in a double strand nucleic acid. The term “blunt end” includesdouble stranded nucleic acid whereby both strands or stretches terminateat the same position, regardless of whether the terminal nucleotide(s)are base paired. In one embodiment, the terminal nucleotide of anantisense strand or antisense stretch and a sense strand or a sensestretch at a blunt end are base paired. In another embodiment, theterminal nucleotide of an antisense strand or an antisense stretch and asense strand or a sense stretch at a blunt end are not paired. Inanother embodiment, the terminal two nucleotides of an antisense strandor an antisense stretch and a sense strand or sense stretch at a bluntend are base paired. In another embodiment, the terminal two nucleotidesof a antisense strand or an antisense stretch and a sense strand orsense stretch at a blunt end are not paired.

In one embodiment, the siRNA has an overhang at one end and a blunt endat the other. In another embodiment, the siRNA has an overhang at bothends. In another embodiment, the siRNA is blunt ended at both ends. Inone embodiment, the siRNA is blunt ended at one end. In anotherembodiment, the siRNA is blunt ended at the end with the 5′-end of theantisense strand or of the antisense stretch and the 3′-end of the sensestrand or of the sense stretch. In another embodiment, the siRNA isblunt ended at the end with the 3′-end of the antisense strand or of theantisense stretch and the 5′-end of the sense strand or of the sensestretch. In another embodiment, the siRNA is blunt ended at both ends.

In another embodiment, the siRNA comprises a overhang at a 3′- or5′-end. In one embodiment, the siRNA has a 3′-overhang on the antisensestrand or on the antisense stretch. In another embodiment, the siRNA hasa 3′-overhang on the sense strand or on the sense stretch. In anotherembodiment, the siRNA has a 5′-overhang on the antisense strand of theantisense stretch. In another embodiment, the siRNA has a 5′-overhang onthe sense strand or at the sense stretch. In another embodiment, thesiRNA has an overhang at both the 5′-end and 3′-end of the antisensestand or of the antisense stretch. In another embodiment, the siRNA hasan overhang at both the 5′-end and 3′-end of the sense stand or of thesense stretch. In another embodiment, the siRNA has a 5′ overhang on theantisense stand or on the antisense stretch and a 3′ overhang on thesense strand or on the sense stretch. In another embodiment, the siRNAhas a 3′ overhang on the antisense stand or on the antisense stretch anda 5′ overhang on the sense strand or on the sense stretch. In anotherembodiment, the siRNA has a 3′ overhang on the antisense stand or on theantisense stretch and a 3′ overhang on the sense strand or on the sensestretch. In another embodiment, the siRNA has a 5′ overhang on theantisense stand or on the antisense stretch and a 5′ overhang on thesense strand or on the sense strand.

In one embodiment, the overhang at the 3 ‘-end of the antisense strandor of the antisense stretch has a length selected from the groupconsisting of 1, 2, 3, 4 and 5 nucleotides. In one embodiment, theoverhang at the 3’-end of the sense strand or of the sense stretch has alength selected from the group consisting of 1, 2, 3, 4 and 5nucleotides. In one embodiment, the overhang at the 5′-end of theantisense strand or of the antisense stretch has a length selected fromthe group consisting of 1, 2, 3, 4 and 5 nucleotides. In one embodiment,the overhang at the 5′-end of the sense strand or of the sense stretchhas a length selected from the group consisting of 1, 2, 3, 4 and 5nucleotides.

Modification:

Another aspect relates to modifications of the siRNA and, in accordancetherewith, the nucleic acid molecule of the invention may be modified asoutlined in the following. It is within the present invention that eachand any modification and pattern disclosed herein, and any disclosureherein related to such modification and pattern, in particular referringto siRNA may also be applicable to and thus realized in connection withand on a nucleic acid molecule of the invention. It is also within thepresent invention that to the extent it is referred to an antisensestrand and/or a sense strand, particularly when it comes to themodification of the antisense strand and/or a sense strand and thenucleotides forming such antisense strand and/or sense strand, suchdisclosure equally applies to an antisense stretch and/or sense stretch,particularly when it comes to the modification of the antisense stretchand/or a sense stretch and the nucleotides forming such antisensestretch and/or sense stretch.

siRNA according to the invention are a ribonucleic acid or a modifiedribonucleic acid. Chemical modifications of the siRNA of the presentinvention provides a powerful tool in overcoming potential limitationsincluding, but not limited to, in vitro and in vivo stability andbioavailability inherent to native RNA molecules. Chemically-modifiedsiRNA can also minimize the possibility of activating interferonactivity in humans. Chemical modification can further enhance thefunctional delivery of a siRNA to a target cell. The modified siRNA ofthe present invention may comprise one or more chemically modifiedribonucleotides of either or both of the antisense strand or the sensestrand. A ribonucleotide may comprise a chemical modification of thebase, sugar or phosphate moieties.

Modifications to Base Moiety:

A secondary aspect relates to modifications to a base moiety. One ormore nucleotides of a siRNA of the present invention may comprise amodified base. A “modified base” means a nucleotide base other than anadenine, guanine, cytosine or uracil at the 1′ position.

In one aspect, the siRNA comprises at least one nucleotide comprising amodified base. In one embodiment, the modified base in on the antisensestrand. In another embodiment, the modified base in on the sense strand.In another embodiment, the modified base is in the duplex region. Inanother embodiment, the modified base is outside the duplex region,i.e., in a single stranded region. In another embodiment, the modifiedbase is on the antisense strand and is outside the duplex region. Inanother embodiment, the modified base is on the sense strand and isoutside the duplex region. In another embodiment, the 3′-terminalnucleotide of the antisense strand is a nucleotide with a modified base.In another embodiment, the 3′-terminal nucleotide of the sense strand isnucleotide with a modified base. In another embodiment, the 5′-terminalnucleotide of the antisense strand is nucleotide with a modified base.In another embodiment, the 5′-terminal nucleotide of the sense strand isnucleotide with a modified base.

In one embodiment, a siRNA has 1 modified base. In another embodiment, asiRNA has about 2-4 modified bases. In another embodiment, a siRNA hasabout 4-6 modified bases. In another embodiment, a siRNA has about 6-8modified bases. In another embodiment, a siRNA has about 8-10 modifiedbases. In another embodiment, a siRNA has about 10-12 modified bases. Inanother embodiment, a siRNA has about 12-14 modified bases. In anotherembodiment, a siRNA has about 14-16 modified bases. In anotherembodiment, a siRNA has about 16-18 modified bases. In anotherembodiment, a siRNA has about 18-20 modified bases. In anotherembodiment, a siRNA has about 20-22 modified bases. In anotherembodiment, a siRNA has about 22-24 modified bases. In anotherembodiment, a siRNA has about 24-26 modified bases. In anotherembodiment, a siRNA has about 26-28 modified bases. In each case thesiRNA comprising said modified bases retains at least 50% of itsactivity as compared to the same siRNA but without said modified bases.

In one embodiment, the modified base is a purine. In another embodiment,the modified base is a pyrimidine. In another embodiment, at least halfof the purines are modified. In another embodiment, at least half of thepyrimidines are modified. In another embodiment, all of the purines aremodified. In another embodiment, all of the pyrimidines are modified.

In another embodiment, the siRNA comprises a nucleotide comprising amodified base, wherein the base is selected from the group consisting of2-aminoadenosine, 2,6-diaminopurine, inosine, pyridin-4-one,pyridin-2-one, phenyl, pseudouracil, 2, 4, 6-trimethoxy benzene,3-methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidine(e.g., 5-methylcytidine), 5-alkyluridine (e.g., ribothymidine),5-halouridine (e.g., 5-bromouridine), 6-azapyrimidine, 6-alkylpyrimidine(e.g. 6-methyluridine), propyne, quesosine, 2-thiouridine,4-thiouridine, wybutosine, wybutoxosine, 4-acetylcytidine,5-(carboxyhydroxymethyl)uridine,5′-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluridine, beta-D-galactosylqueosine,1-methyladenosine, 1-methylinosine, 2,2-dimethylguanosine,3-methylcytidine, 2-methyladenosine, 2-methylguanosine,N6-methyladenosine, 7-methylguanosine,5-methoxyaminomethyl-2-thiouridine, 5-methylaminomethyluridine,5-methylcarbonylmethyluridine, 5-methyloxyuridine,5-methyl-2-thiouridine, 2-methylthio-N6-isopentenyladenosine,beta-D-mannosylqueosine, uridine-5-oxyacetic acid and 2-thiocytidine.

In another aspect, a siRNA of the present invention comprises an abasicnucleotide. The term “abasic” as used herein, refers to moieties lackinga base or having other chemical groups in place of a base at the 1′position, for example a 3′,3′-linked or 5′,5′-linked deoxyabasic ribosederivative. As used herein, a nucleotide with a modified base does notinclude abasic nucleotides. In one aspect, the siRNA comprises at leastone abasic nucleotide. In one embodiment, the abasic nucleotide is onthe antisense strand. In another embodiment, the abasic nucleotide is onthe sense strand. In another embodiment, the abasic nucleotide is in theduplex region. In another embodiment, the abasic nucleotide is outsidethe duplex region. In another embodiment, the abasic nucleotide is onthe antisense strand and is outside the duplex region. In anotherembodiment, the abasic nucleotide is on the sense strand and is outsidethe duplex region. In another embodiment, the 3′-terminal nucleotide ofthe antisense strand is an abasic nucleotide. In another embodiment, the3′-terminal nucleotide of the sense strand is an abasic nucleotide. Inanother embodiment, the 5′-terminal nucleotide of the antisense strandis an abasic nucleotide. In another embodiment, the 5′-terminalnucleotide of the sense strand is an abasic nucleotide. In anotherembodiment, a siRNA has a number of abasic nucleotides selected from thegroup consisting of 1, 2, 3, 4, 5 and 6.

Modifications to Sugar Moiety:

Another secondary aspect relates to modifications to a sugar moiety. Oneor more nucleotides of an siRNA of the present invention may comprise amodified ribose moiety.

Modifications at the 2′-position wherein the 2′-OH is substitutedinclude the non-limiting examples selected from the group consisting ofalkyl, substituted alkyl, alkaryl-, aralkyl-, —F, —Cl, —Br, —CN, —CF3,—OCF3, —OCN, —O-alkyl, —S-alkyl, HS-alkyl-O, —O-alkenyl, —S-alkenyl,—N-alkenyl, —SO-alkyl, -alkyl-OSH, -alkyl-OH, —O-alkyl-OH, —O-alkyl-SH,—S-alkyl-OH, —S— alkyl-SH, -alkyl-S-alkyl, -alkyl-O-alkyl, —ONO2, —NO2,—N3, —NH2, alkylamino, dialkylamino-, aminoalkyl-, aminoalkoxy,aminoacid, aminoacyl-, —ONH2, —O-aminoalkyl, —O-aminoacid, —O-aminoacyl,heterocycloalkyl-, heterocycloalkaryl-, aminoalkylamino-,polyalklylamino-, substituted silyl-, methoxyethyl-(MOE), alkenyl andalkynyl. “Locked” nucleic acids (LNA) in which the 2′ hydroxyl isconnected, e.g., by a methylene bridge, to the 4′ carbon of the sameribose sugar is further included as a 2′ modification of the presentinvention. Preferred substitutents are 2′-methoxyethyl, 2′-OCH3,2′-O-allyl, 2′-C-allyl, and 2′-fluoro.

In one embodiment, the siRNA comprises 1-5 2′-modified nucleotides. Inanother embodiment, the siRNA comprises 5-10 2′-modified nucleotides. Inanother embodiment, the siRNA comprises 15-20 2′-modified nucleotides.In another embodiment, the siRNA comprises 20-25 2′-modifiednucleotides. In another embodiment, the siRNA comprises 25-302′-modified nucleotides.

In one embodiment, the antisense strand comprises 1-2 2′-modifiednucleotides. In one embodiment, the antisense strand comprises about 2-42′-modified nucleotides. In one embodiment, the antisense strandcomprises about 4-6 2′-modified nucleotides. In one embodiment, theantisense strand comprises about 6-8 2′-modified nucleotides. In oneembodiment, the antisense strand comprises about 8-10 2′-modifiednucleotides. In one embodiment, the antisense strand comprises about10-12 2′-modified nucleotides. In one embodiment, the antisense strandcomprises about 12-14 2′-modified nucleotides. In one embodiment, theantisense strand comprises about 14-16 2′-modified nucleotides. In oneembodiment, the antisense strand comprises about 16-18 2′-modifiednucleotides. In one embodiment, the antisense strand comprises about18-20 2′-modified nucleotides. In one embodiment, the antisense strandcomprises about 22-24 2′-modified nucleotides. In one embodiment, theantisense strand comprises about 24-26 2′-modified nucleotides.

In one embodiment, the sense strand comprises 1-2 2′-modifiednucleotides. In one embodiment, the sense strand comprises about 2-42′-modified nucleotides. In one embodiment, the sense strand comprisesabout 4-6 2′-modified nucleotides. In one embodiment, the sense strandcomprises about 6-8 2′-modified nucleotides. In one embodiment, thesense strand comprises about 8-10 2′-modified nucleotides. In oneembodiment, the sense strand comprises about 10-12 2′-modifiednucleotides. In one embodiment, the sense strand comprises about 12-142′-modified nucleotides. In one embodiment, the sense strand comprisesabout 14-16 2′-modified nucleotides. In one embodiment, the sense strandcomprises about 16-18 2′-modified nucleotides. In one embodiment, thesense strand comprises about 18-20 2′-modified nucleotides. In oneembodiment, the sense strand comprises about 22-24 2′-modifiednucleotides. In one embodiment, the sense strand comprises about 24-262′-modified nucleotides.

In one embodiment, the siRNA comprises 1-5 2′-OCH3 modified nucleotides.In another embodiment, the siRNA comprises 5-10 2′-OCH3 modifiednucleotides. In another embodiment, the siRNA comprises 15-20 2′-OCH3modified nucleotides. In another embodiment, the siRNA comprises 20-252′-OCH3 modified nucleotides. In another embodiment, the siRNA comprises25-30 2′-OCH3 modified nucleotides.

In one embodiment, the antisense strand comprises 1-2 2′-OCH3 modifiednucleotides. In one embodiment, the antisense strand comprises about 2-42′-OCH3 modified nucleotides. In one embodiment, the antisense strandcomprises about 4-6 2′-OCH3 modified nucleotides. In one embodiment, theantisense strand comprises about 6-8 2′-OCH3 modified nucleotides. Inone embodiment, the antisense strand comprises about 8-10 2′-OCH3modified nucleotides. In one embodiment, the antisense strand comprisesabout 10-12 2′-OCH3 modified nucleotides.

In one embodiment, the antisense strand comprises about 12-14 2′-OCH3modified nucleotides. In one embodiment, the antisense strand comprisesabout 14-16 2′-OCH3 modified nucleotides. In one embodiment, theantisense strand comprises about 16-18 2′-OCH3 modified nucleotides. Inone embodiment, the antisense strand comprises about 18-20 2′-OCH3modified nucleotides. In one embodiment, the antisense strand comprisesabout 22-24 2′-OCH3 modified nucleotides. In one embodiment, theantisense strand comprises about 24-26 2′-OCH3 modified nucleotides.

In one embodiment, the sense strand comprises 1-2 2′-OCH3 modifiednucleotides. In one embodiment, the sense strand comprises about 2-42′-OCH3 modified nucleotides. In one embodiment, the sense strandcomprises about 4-6 2′-OCH3 modified nucleotides. In one embodiment, thesense strand comprises about 6-8 2′-OCH3 modified nucleotides. In oneembodiment, the sense strand comprises about 8-10 2′-OCH3 modifiednucleotides. In one embodiment, the sense strand comprises about 10-122′-OCH3 modified nucleotides. In one embodiment, the sense strandcomprises about 12-14 2′-OCH3 modified nucleotides. In one embodiment,the sense strand comprises about 14-16 2′-OCH3 modified nucleotides. Inone embodiment, the sense strand comprises about 16-18 2′-OCH3 modifiednucleotides. In one embodiment, the sense strand comprises about 18-202′-OCH3 modified nucleotides. In one embodiment, the sense strandcomprises about 22-24 2′-OCH3 modified nucleotides. In one embodiment,the sense strand comprises about 24-26 2′-OCH3 modified nucleotides.

In one embodiment, the siRNA duplex region comprises 1-5 2′-OCH3modified nucleotides. In another embodiment, the siRNA duplex regioncomprises 5-10 2′-OCH3 modified nucleotides. In another embodiment, thesiRNA duplex region comprises 15-20 2′-OCH3 modified nucleotides. Inanother embodiment, the siRNA duplex region comprises 20-25 2′-OCH3modified nucleotides. In another embodiment, the siRNA duplex regioncomprises 25-30 2′-OCH3 modified nucleotides.

In one embodiment, the antisense duplex region comprises 1-2 2′-OCH3modified nucleotides. In one embodiment, the antisense duplex regioncomprises about 2-4 2′-OCH3 modified nucleotides. In one embodiment, theantisense duplex region comprises about 4-6 2′-OCH3 modifiednucleotides. In one embodiment, the antisense duplex region comprisesabout 6-8 2′-OCH3 modified nucleotides. In one embodiment, the antisenseduplex region comprises about 8-10 2′-OCH3 modified nucleotides. In oneembodiment, the antisense duplex region comprises about 10-12 2′-OCH3modified nucleotides. In one embodiment, the antisense duplex regioncomprises about 12-14 2′-OCH3 modified nucleotides. In one embodiment,the antisense duplex region comprises about 14-16 2′-OCH3 modifiednucleotides. In one embodiment, the antisense duplex region comprisesabout 16-18 2′-OCH3 modified nucleotides. In one embodiment, theantisense duplex region comprises about 18-20 2′-OCH3 modifiednucleotides. In one embodiment, the antisense duplex region comprisesabout 22-24 2′-OCH3 modified nucleotides. In one embodiment, theantisense duplex region comprises about 24-26 2′-OCH3 modifiednucleotides.

In one embodiment, the sense duplex region comprises 1-2 2′-OCH3modified nucleotides. In another embodiment, the sense duplex regioncomprises about 2-4 2′-OCH3 modified nucleotides. In another embodiment,the sense duplex region comprises about 4-6 2′-OCH3 modifiednucleotides. In another embodiment, the sense duplex region comprisesabout 6-8 2′-OCH3 modified nucleotides. In another embodiment, the senseduplex region comprises about 8-10 2′-OCH3 modified nucleotides. Inanother embodiment, the sense duplex region comprises about 10-122′-OCH3 modified nucleotides. In another embodiment, the sense duplexregion comprises about 12-14 2′-OCH3 modified nucleotides. In anotherembodiment, the sense duplex region comprises about 14-16 2′-OCH3modified nucleotides. In another embodiment, the sense duplex regioncomprises about 16-18 2′-OCH3 modified nucleotides. In anotherembodiment, the sense duplex region comprises about 18-20 2′-OCH3modified nucleotides. In another embodiment, the sense duplex regioncomprises about 22-24 2′-OCH3 modified nucleotides. In anotherembodiment, the sense duplex region comprises about 24-26 2′-OCH3modified nucleotides.

In one embodiment, the siRNA comprises an antisense strand 19nucleotides in length and a sense strand 19 nucleotides in length,wherein said antisense strand comprises 2′-OCH3 modifications atnucleotides 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19, and wherein said sensestrand comprises 2′-OCH3 modifications at nucleotides 2, 4, 6, 8, 10,12, 14, 16 and 18, wherein said antisense strand is numbered from 5′-3′and said sense strand is numbered from 3′-5′. In another embodiment, thesiRNA comprises an antisense strand 20 nucleotides in length and a sensestrand 20 nucleotides in length, wherein said antisense strand comprises2′-OCH3 modifications at nucleotides 1, 3, 5, 7, 9, 11, 13, 15, 17 and19, and wherein said sense strand comprises 2′-OCH3 modifications atnucleotides 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 wherein said antisensestrand is numbered from 5′-3′ and said sense strand is numbered from3′-5′. In another embodiment, the siRNA comprises an antisense strand 21nucleotides in length and a sense strand 21 nucleotides in length,wherein said antisense strand comprises 2′-OCH3 modifications atnucleotides 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 and 21, and wherein saidsense strand comprises 2′-OCH3 modifications at nucleotides 2, 4, 6, 8,10, 12, 14, 16, 18 and 20, wherein said antisense strand is numberedfrom 5′-3′ and said sense strand is numbered from 3′-5′. In anotherembodiment, the siRNA comprises an antisense strand 22 nucleotides inlength and a sense strand 22 nucleotides in length, wherein saidantisense strand comprises 2′-OCH3 modifications at nucleotides 1, 3, 5,7, 9, 11, 13, 15, 17, 19 and 21, and wherein said sense strand comprises2′-OCH3 modifications at nucleotides 2, 4, 6, 8, 10, 12, 14, 16, 18, 20and 22, wherein said antisense strand is numbered from 5′-3′ and saidsense strand is numbered from 3′-5′. In another embodiment, the siRNAcomprises an antisense strand 23 nucleotides in length and a sensestrand 23 nucleotides in length, wherein said antisense strand comprises2′-OCH3 modifications at nucleotides 1, 3, 5, 7, 9, 11, 13, 15, 17, 19,21 and 23, and wherein said sense strand comprises 2′-OCH3 modificationsat nucleotides 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 and 22 wherein saidantisense strand is numbered from 5′-3′ and said sense strand isnumbered from 3′-5′.

In another embodiment, the siRNA comprises an antisense strand 18-23nucleotides in length and a sense strand 18-23 nucleotides in length,wherein said antisense strand comprises 2′-OCH3 modifications atnucleotides 3, 5, 7, 9, 11, 13, 15 and 17, and wherein said sense strandcomprises 2′-OCH3 modifications at nucleotides 4, 6, 8, 10, 12, 14 and16, wherein said antisense strand is numbered from 5′-3′ and said sensestrand is numbered from 3′-5′. In another embodiment, the siRNAcomprises an antisense strand 18-23 nucleotides in length and a sensestrand 18-23 nucleotides in length, wherein said antisense strandcomprises 2′-OCH3 modifications at nucleotides 5, 7, 9, 11, 13 and 15,and wherein said sense strand comprises 2′-OCH3 modifications atnucleotides 6, 8, 10, 12 and 14, wherein said antisense strand isnumbered from 5′-3′ and said sense strand is numbered from 3′-5′. Inanother embodiment, the siRNA comprises an antisense strand 18-23nucleotides in length and a sense strand 18-23 nucleotides in length,wherein said antisense strand comprises 2′-OCH3 modifications atnucleotides 7, 9, 11, 13 and wherein said sense strand comprises 2′-OCH3modifications at nucleotides 8, 10 and 12, wherein said antisense strandis numbered from 5′-3′ and said sense strand is numbered from 3′-5′. Inanother embodiment, the siRNA comprises an antisense strand 18-23nucleotides in length and a sense strand 18-23 nucleotides in length,wherein said antisense strand comprises 2′-OCH3 modifications atnucleotides 7, 9 and 11, and wherein said sense strand comprises 2′-OCH3modifications at nucleotides 8, 10 and 12, wherein said antisense strandis numbered from 5′-3′ and said sense strand is numbered from 3′-5′. Inanother embodiment, the siRNA comprises an antisense strand 18-23nucleotides in length and a sense strand 18-23 nucleotides in length,wherein said antisense strand comprises 2′-OCH3 modifications atnucleotides 7 and 9, and wherein said sense strand comprises 2′-OCH3modifications at nucleotides 8 and 10, wherein said antisense strand isnumbered from 5′-3′ and said sense strand is numbered from 3′-5′. Inanother embodiment, the siRNA comprises an antisense strand 18-23nucleotides in length and a sense strand 18-23 nucleotides in length,wherein said antisense strand comprises 2′-OCH3 modifications atnucleotides 9 and 11, and wherein said sense strand comprises 2′-OCH3modifications at nucleotides 8 and 10, wherein said antisense strand isnumbered from 5′-3′ and said sense strand is numbered from 3′-5′.

In further embodiments, the siRNA comprises the following nucleotidesequences, wherein the sequences comprise 2′-OCH3 modifications onnucleotides indicated with a capital letter:

In another embodiment, the antisense strand comprises 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or25 2′-deoxy nucleotides.

In another embodiment, the sense strand comprises 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 252′-deoxy nucleotides.

In another embodiment, the antisense strand comprises 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or25 2′-fluoro nucleotides.

In another embodiment, the sense strand comprises 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 252′-fluoro nucleotides.

In another embodiment, the pyrimidine nucleotides in the antisensestrand are 2′-O-methyl pyrimidine nucleotides.

In another embodiment, of the purine nucleotides in the antisense strandare 2′-O-methyl purine nucleotides.

In another embodiment, the pyrimidine nucleotides in the antisensestrand are 2′-deoxy pyrimidine nucleotides.

In another embodiment, the purine nucleotides in the antisense strandare 2′-deoxy purine nucleotides.

In another embodiment, the pyrimidine nucleotides in the antisensestrand are 2′-fluoro pyrimidine nucleotides.

In another embodiment, the purine nucleotides in the antisense strandare 2′-fluoro purine nucleotides.

In another embodiment, the pyrimidine nucleotides in the sense strandare 2′-O-methyl pyrimidine nucleotides.

In another embodiment, of the purine nucleotides in the sense strand are2′-O-methyl purine nucleotides.

In another embodiment, the pyrimidine nucleotides in the sense strandare 2′-deoxy pyrimidine nucleotides.

In another embodiment, the purine nucleotides in the sense strand are2′-deoxy purine nucleotides.

In another embodiment, the pyrimidine nucleotides in the sense strandare 2′-fluoro pyrimidine nucleotides.

In another embodiment, the purine nucleotides in the sense strand are2′-fluoro purine nucleotides.

In another embodiment, the pyrimidine nucleotides in the antisenseduplex region are 2′-O-methyl pyrimidine nucleotides.

In another embodiment, of the purine nucleotides in the antisense duplexregion are 2′-O-methyl purine nucleotides.

In another embodiment, the pyrimidine nucleotides in the antisenseduplex region are 2′-deoxy pyrimidine nucleotides.

In another embodiment, the purine nucleotides in the antisense duplexregion are 2′-deoxy purine nucleotides.

In another embodiment, the pyrimidine nucleotides in the antisenseduplex region are 2′-fluoro pyrimidine nucleotides.

In another embodiment, the purine nucleotides in the antisense duplexregion are 2′-fluoro purine nucleotides.

In another embodiment, the pyrimidine nucleotides in the sense duplexregion are 2′43-methyl pyrimidine nucleotides.

In another embodiment, of the purine nucleotides in the sense duplexregion are 2′-O-methyl purine nucleotides.

In another embodiment, the pyrimidine nucleotides in the sense duplexregion are 2′-deoxy pyrimidine nucleotides.

In another embodiment, the purine nucleotides in the sense duplex regionare 2′-deoxy purine nucleotides.

In another embodiment, the pyrimidine nucleotides in the sense duplexregion are 2′-fluoro pyrimidine nucleotides.

In another embodiment, the purine nucleotides in the sense duplex regionare 2′-fluoro purine nucleotides.

In another embodiment, the pyrimidine nucleotides in the antisenseduplex flanking regions are 2′-O-methyl pyrimidine nucleotides.

In another embodiment, of the purine nucleotides in the antisense duplexflanking regions are 2′-O-methyl purine nucleotides.

In another embodiment, the pyrimidine nucleotides in the antisenseduplex flanking regions are 2′-deoxy pyrimidine nucleotides.

In another embodiment, the purine nucleotides in the antisense duplexflanking regions are 2′-deoxy purine nucleotides.

In another embodiment, the pyrimidine nucleotides in the antisenseduplex flanking regions are 2′-fluoro pyrimidine nucleotides.

In another embodiment, the purine nucleotides in the antisense duplexflanking regions are 2′-fluoro purine nucleotides.

In another embodiment, the pyrimidine nucleotides in the sense duplexflanking regions are 2′-O-methyl pyrimidine nucleotides.

In another embodiment, of the purine nucleotides in the sense duplexflanking regions are 2′-O-methyl purine nucleotides.

In another embodiment, the pyrimidine nucleotides in the sense duplexflanking regions are 2′-deoxy pyrimidine nucleotides.

In another embodiment, the purine nucleotides in the sense duplexflanking regions are 2′-deoxy purine nucleotides.

In another embodiment, the pyrimidine nucleotides in the sense duplexflanking regions are 2′-fluoro pyrimidine nucleotides.

In another embodiment, the purine nucleotides in the sense duplexflanking regions are 2′-fluoro purine nucleotides.

Pattern: It is within the present invention that any disclosure relatedto pattern provided herein specifically referring to an antisense strandequally applies to an antisense stretch, and any disclosure related topattern provided herein specifically referring to an sense strandequally applies to a sense stretch

In one embodiment, the antisense duplex region comprises a plurality ofgroups of modified nucleotides, referred to herein as “modified groups”,wherein each modified group consists of one or more identically modifiednucleotides, wherein each modified group is flanked on one or both sidesby a second group of nucleotides, referred to herein as “flankinggroups”, wherein each said flanking group consists of one or morenucleotides that are either unmodified or modified in a manner differentfrom the nucleotides of said modified group. In one embodiment, eachmodified group in the antisense duplex region is identical, i.e., eachmodified group consists of an equal number of identically modifiednucleotides. In another embodiment, each flanking group has an equalnumber of nucleotide. In another embodiment, each flanking group isidentical. In another embodiment, the nucleotides of said modifiedgroups in the antisense duplex region comprise a modified base. Inanother embodiment, the nucleotides of said modified groups comprise amodified phosphate backbone. In another embodiment, the nucleotides ofsaid modified groups comprise a modified 2′ position.

In another aspect, the sense duplex region comprises a plurality ofgroups of modified groups, wherein each modified group consists of oneor more identically modified nucleotides, wherein each modified group isflanked on one or both sides by a flanking group, wherein each saidflanking group consists of one or more nucleotides that are eitherunmodified or modified in a manner different from the nucleotides ofsaid modified group. In one embodiment, each modified group in the senseduplex region is identical. In another embodiment, each flanking grouphas an equal number of nucleotides. In another embodiment, each flankinggroup is identical. In another embodiment, the nucleotides of saidmodified groups in the sense duplex region comprise a modified base. Inanother embodiment, the nucleotides of said modified groups comprise amodified phosphate backbone. In another embodiment, the nucleotides ofsaid modified groups comprise a modified 2′ position.

In another aspect, the antisense duplex region and the sense duplexregion each comprise a plurality of modified groups, wherein eachmodified group consists of one or more identically modified nucleotides,wherein each modified group is flanked on one or both sides by aflanking group, wherein each said flanking group consists of one or morenucleotides that are either unmodified or modified in a manner differentfrom the nucleotides of said modified group. In one embodiment, eachmodified group in the antisense duplex region and the sense duplexregion are identical. In another embodiment, each flanking group in theantisense duplex region and the sense duplex region has an equal numberof nucleotides. In another embodiment, each flanking group in theantisense duplex region and in the sense duplex region are identical. Inanother embodiment, the nucleotides of said modified groups in theantisense duplex region and the sense duplex region each comprise thesame modified groups and the same flanking groups. In anotherembodiment, the nucleotides of said modified groups in the antisenseduplex region and the sense duplex region each comprise a modified base.In another embodiment, the nucleotides of said modified groups in theantisense duplex region and the sense duplex region each comprise amodified phosphate backbone. In another embodiment, the nucleotides ofsaid modified groups in the antisense duplex region and the sense duplexregion each comprise a modified 2′ position.

In one aspect, the antisense strand comprises a plurality of groups ofmodified nucleotides, referred to herein as “modified groups”, whereineach modified group consists of one or more identically modifiednucleotides, wherein each modified group is flanked on one or both sidesby a second group of nucleotides, referred to herein as “flankinggroups”, wherein each said flanking group consists of one or morenucleotides that are either unmodified or modified in a manner differentfrom the nucleotides of said modified group. In one embodiment, eachmodified group in the antisense strand is identical, i.e., each modifiedgroup consists of an equal number of identically modified nucleotides.In another embodiment, each flanking group has an equal number ofnucleotide. In another embodiment, each flanking group is identical. Inanother embodiment, the nucleotides of said modified groups in theantisense strand comprise a modified base. In another embodiment, thenucleotides of said modified groups comprise a modified phosphatebackbone. In another embodiment, the nucleotides of said modified groupscomprise a modified 2′ position.

In another aspect, the sense strand comprises a plurality of groups ofmodified groups, wherein each modified group consists of one or moreidentically modified nucleotides, wherein each modified group is flankedon one or both sides by a flanking group, wherein each said flankinggroup consists of one or more nucleotides that are either unmodified ormodified in a manner different from the nucleotides of said modifiedgroup. In one embodiment, each modified group in the sense strand isidentical. In another embodiment, each flanking group has an equalnumber of nucleotides. In another embodiment, each flanking group isidentical. In another embodiment, the nucleotides of said modifiedgroups in the sense strand comprise a modified base. In anotherembodiment, the nucleotides of said modified groups comprise a modifiedphosphate backbone. In another embodiment, the nucleotides of saidmodified groups comprise a modified 2′ position.

In another aspect, the antisense strand and the sense strand eachcomprise a plurality of modified groups, wherein each modified groupconsists of one or more identically modified nucleotides, wherein eachmodified group is flanked on one or both sides by a flanking group,wherein each said flanking group consists of one or more nucleotidesthat are either unmodified or modified in a manner different from thenucleotides of said modified group. In one embodiment, each modifiedgroup in the antisense strand and the sense strand are identical. Inanother embodiment, each flanking group in the antisense strand and thesense strand each have an equal number of nucleotides. In anotherembodiment, each flanking group in the antisense strand and in the sensestrand are identical. In another embodiment, the nucleotides of saidmodified groups in the antisense strand and the sense strand eachcomprise the same modified groups and the same flanking groups. Inanother embodiment, the nucleotides of said modified groups in theantisense strand and the sense strand each comprise a modified base. Inanother embodiment, the nucleotides of said modified groups in theantisense strand and the sense strand each comprise a modified phosphatebackbone. In another embodiment, the nucleotides of said modified groupsin the antisense strand and the sense strand each comprise a modified 2′position.

In another aspect, the modified groups and the flanking groups form aregular pattern on the antisense stand. In another aspect, the modifiedgroups and the flanking groups form a regular pattern on the sensestrand. In one embodiment, the modified groups and the flanking groupsform a regular pattern on the both the antisense strand and the sensestrand. In another embodiment, the modified groups and the flankinggroups form a regular pattern on the antisense duplex region. In anotheraspect, the modified groups and the flanking groups form a regularpattern on the sense duplex region. In one embodiment, the modifiedgroups and the flanking groups form a regular pattern on the both theantisense duplex region and the sense duplex region.

In another aspect, the pattern is a spatial or positional pattern. Aspatial or positional pattern means that (a) nucleotide(s) are modifieddepending on their position within the nucleotide sequence of adouble-stranded portion. Accordingly, it does not matter whether thenucleotide to be modified is a pyrimidine or a purine. Rather theposition of a modified nucleotide is dependent upon: (a) its numberedposition on a strand of nucleic acid, wherein the nucleotides arenumbered from the 5′-end to the 3′-end with the 5′-end nucleotide of thestrand being position one (both the antisense strand and sense strandare numbered from their respective 5′-end nucleotide), or (b) theposition of the modified group relative to a flanking group. Thus,according to this embodiment, the modification pattern will always bethe same, regardless of the sequence which is to be modified.

In another embodiment, the number of modified groups on the antisensestrand is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14.In another embodiment, the number of modified groups on the sense strandis selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14. Inanother embodiment, the number of flanking groups on the antisensestrand of nucleic acid is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13 or 14. In another embodiment, the number of flanking groupson the sense strand of nucleic acid is selected from 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13 or 14. In one embodiment, the number of modifiedgroups and the number of flanking groups on either or both the antisensestrand and the sense strand are the same.

In another embodiment, the number of modified groups on the antisenseduplex region is selected 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or14. In another embodiment, the number of modified groups on the senseduplex region is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13or 14. In another embodiment, the number of flanking groups on theantisense duplex region of nucleic acid is 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13 or 14. In another embodiment, the number of flankinggroups on the sense duplex region of nucleic acid is selected from 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14. In one embodiment, the numberof modified groups and the number of flanking groups on either or boththe antisense duplex region and the sense duplex region are the same.

In one embodiment, the number of modified groups and the number offlanking groups on a strand or on a duplex region are the same. Inanother embodiment, the number of modified groups and the number offlanking groups on a strand or on a duplex region are the same, whereinthe number is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or14.

In another embodiment, the number of nucleotides in a modified group isselected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14. Inanother embodiment, the number of nucleotides in a flanking group isselected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14.

In one embodiment, each modified group on both the antisense strand andthe sense strand is identical. In one embodiment, each modified group onboth the antisense duplex region and the sense duplex region isidentical. In another embodiment, each modified group and each flankinggroup on both the antisense strand and the sense strand are identical.In one embodiment, each modified group and each flanking group on boththe antisense duplex region and the sense duplex region are identical.

In one embodiment, each modified group, each modified group position,each flanking group and each flanking group position on both theantisense strand and the sense strand are identical. In one embodiment,each modified group, each modified group position, each flanking groupand each flanking group position on both the antisense duplex region andthe sense duplex region are identical. In another embodiment, themodified groups on the antisense strand are complementary with themodified groups on the sense strand (the modified groups on theantisense strand and the sense strand are perfectly aligned across fromone another). In another embodiment, there are no mismatches in themodified groups such that each modified group on the antisense strand isbase paired with each modified group on the sense strand. In anotherembodiment, each modified group on the sense strand is shifted by 1, 2,3, 4 or 5 nucleotides relative to the modified groups on the antisensestrand. For example, if each modified group on the sense strand isshifted by one nucleotide and a modified group started at position oneon the antisense strand, a modified group on the sense strand wouldbegin at position two. In another embodiment, the modified groups of theantisense strand do not overlap the modified groups of the sense strand,i.e., no nucleotide of a modified group on the antisense strand is basepaired with a nucleotide of a modified group on the sense strand.

In one embodiment, deoxyribonucleotides at an end of a strand of nucleicacid are not considered when determining a position of a modified group,i.e., the positional numbering begins with the first ribonucleotide ormodified ribonucleotide. In another embodiment, abasic nucleotides at anend of a strand of nucleic acid are not considered when determining aposition of a modified group.

In one aspect, a modified group comprises a 5′-end nucleotide of eitheror both of the antisense strand and the sense strand. In anotherembodiment, a flanking group comprises the 5′-end nucleotide of eitheror both of the antisense strand and the sense strand. In anotherembodiment, the 5′-end nucleotide of either or both of the antisensestrand and the sense strand is unmodified. In another embodiment, amodified group comprises the 5′-most nucleotide of either or both of theantisense duplex region and sense duplex region. In another embodiment,a flanking group comprises the 5′-most nucleotide of either or both ofthe antisense duplex region or the sense duplex region. In anotherembodiment, the 5′-most nucleotide of either or both of the antisenseduplex region or the sense duplex region is unmodified. In anotherembodiment, the nucleotide at position 10 of the antisense strand isunmodified. In another embodiment, the nucleotide at position 10 of thesense strand is modified. In another embodiment, a modified groupcomprises the nucleotide at position 10 of the sense strand.

In one embodiment, the modification at the 2′ position is selected fromthe group comprising amino, fluoro, methoxy, alkoxy and C₁-C₃-alkyl. Inanother embodiment, the modification is 2′-O-methyl.

In another aspect, each modified group consists of one nucleotide andeach flanking group consists of one nucleotide. In one embodiment, eachmodified group on the antisense strand is aligned with a flanking groupon the sense strand.

In another aspect, each modified group consists of one 2′-O-methylmodified nucleotide and each flanking group consists of one nucleotide.In one embodiment, each flanking group consists of one unmodifiednucleotide. In one embodiment, each flanking group consists of one2′-O-methyl modified nucleotide. In another embodiment, each modifiedgroup on both the antisense strand and the sense strand consists of one2′-O-methyl modified nucleotide and each flanking group on both theantisense strand and the sense strand consists of one nucleotide,wherein no modified group on one strand is either aligned or bothaligned and base paired with another modified group on the other strandand no flanking group on one strand is either aligned or both alignedand base paired with a flanking group on the other strand. In anotherembodiment, excluding any optional overhangs, each modified group oneach strand is either aligned or both aligned and based paired with aflanking group on the other strand. In one embodiment, the flankinggroup is unmodified. In another embodiment, the nucleotide of positionone on the antisense strand is 2′-O-methyl modified. In anotherembodiment, the 5′-most nucleotide of the antisense duplex region is2′-O-methyl modified.

Positional modification schemes are described in international patentapplication WO 2004/015107, incorporated by reference in its entirety.

Modifications to Phosphate Backbone:

It is within the present invention that any disclosure related tomodification to phosphate backbone provided herein specificallyreferring to an antisense strand or nucleotides forming such antisensestrand equally applies to an antisense stretch or nucleotides formingsuch antisense stretch, and any disclosure related to modification tophosphate backbone provided herein specifically referring to a sensestrand or nucleotides forming such sense strand equally applies to asense stretch or nucleotides forming such sense stretch.

In one embodiment, the nucleic acid molecule of the invention and thesiRNA of the invention in particular bear, have or display one orseveral modifications to a phosphate backbone, whereby such modificationis preferably one described herein.

All or a portion of the nucleotides of the siRNA of the invention may belinked through phosphodiester bonds, as found in unmodified nucleicacid. A siRNA of the present invention however, may comprise a modifiedphosphodiester linkage. The phosphodiester linkages of either theantisense stand or the sense strand may be modified to independentlyinclude at least one heteroatom selected from the group consisting ofnitrogen and sulfur. In one embodiment, a phosphoester group connectinga ribonucleotide to an adjacent ribonucleotide is replaced by a modifiedgroup. In one embodiment, the modified group replacing the phosphoestergroup is selected from the group consisting of phosphothioate,methylphosphonate or phosphoramidate group.

In one embodiment, all of the nucleotides of the antisense strand arelinked through phosphodiester bonds. In another embodiment, all of thenucleotides of the antisense duplex region are linked throughphosphodiester bonds. In another embodiment, all of the nucleotides ofthe sense strand are linked through phosphodiester bonds. In anotherembodiment, all of the nucleotides of the sense duplex region are linkedthrough phosphodiester bonds. In another embodiment, the antisensestrand comprises a number of modified phosphodiester groups selectedfrom 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, theantisense duplex region comprises a number of modified phosphodiestergroups selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In anotherembodiment, the sense strand comprises a number of modifiedphosphodiester groups selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. Inanother embodiment, the sense duplex region comprises a number ofmodified phosphodiester groups selected from 1, 2, 3, 4, 5, 6, 7, 8, 9or 10.

In another embodiment, one or more nucleotides forming the antisenseduplex region, the sense duplex region or the overhang(s) are linkedthrough phosphorothioate bonds. In a preferred embodiment, thenucleotides forming the overhang are linked to each other by one or morephosphorothioate bonds.

5′ and 3′ End Modifications:

It is within the present invention that any disclosure related to 5′ and3′ end modifications provided herein specifically referring to anantisense strand or nucleotides forming such antisense strand equallyapplies to an antisense stretch or nucleotides forming such antisensestretch, and any disclosure related to 5′ and 3′ end modificationsmodification provided herein specifically referring to a sense strand ornucleotides forming such sense strand equally applies to a sense stretchor nucleotides forming such sense stretch.

In one embodiment, the nucleic acid molecule of the invention and thesiRNA of the invention in particular bear, have or display a 5′ and/or3′ modification, whereby such modification is preferably one describedherein.

The siRNA of the present invention may include nucleic acid moleculescomprising one or more modified nucleotides, abasic nucleotides, acyclicor deoxyribonucleotide at the terminal 5′- or 3′-end on either or bothof the sense or antisense strands. In one embodiment, the 5′- and 3′-endnucleotides of both the sense and antisense strands are unmodified. Inanother embodiment, the 5′-end nucleotide of the antisense strand ismodified. In another embodiment, the 5′-end nucleotide of the sensestrand is modified. In another embodiment, the 3′-end nucleotide of theantisense strand is modified. In another embodiment, the 3′-endnucleotide of the sense strand is modified. In another embodiment, the5′-end nucleotide of the antisense strand and the 5′-end nucleotide ofthe sense strand are modified. In another embodiment, the 3′-endnucleotide of the antisense strand and the 3′-end nucleotide of thesense strand are modified. In another embodiment, the 5′-end nucleotideof the antisense strand and the 3′-end nucleotide of the sense strandare modified. In another embodiment, the 3′-end nucleotide of theantisense strand and the 5′-end nucleotide of the sense strand aremodified. In another embodiment, the 3′-end nucleotide of the antisensestrand and both the 5′- and 3′-end nucleotides of the sense strand aremodified. In another embodiment, both the 5′- and 3′-end nucleotides ofthe antisense strand are modified. In another embodiment, both the 5′-and 3′-end nucleotides of the sense strand are modified.

In another embodiment, the 5′-end nucleotide of the antisense strand isphosphorylated. In another embodiment, the 5′-end nucleotide of thesense strand is phosphorylated. In another embodiment, the 5′-endnucleotides of both the antisense strand and the sense strand arephosphorylated. In another embodiment, the 5′-end nucleotide of theantisense strand is phosphorylated and the 5′-end nucleotide of thesense strand has a free hydroxyl group (5′-OH). In another embodiment,the 5′-end nucleotide of the antisense strand is phosphorylated and the5′-end nucleotide of the sense strand is modified.

Modifications to the 5′- and 3′-end nucleotides are not limited to the5′ and 3′ positions on these terminal nucleotides. Examples ofmodifications to end nucleotides include, but are not limited to,biotin, inverted (deoxy) abasics, amino, fluoro, chloro, bromo, CN, CF,methoxy, imidazole, caboxylate, thioate, C1 to C10 lower alkyl,substituted lower alkyl, alkaryl or aralkyl, OCF3, OCN, O-, S-, orN-alkyl; O-, S-, or N-alkenyl; SOCH3; SO2CH3; ONO2; NO2, N3;heterozycloalkyl; heterozycloalkaryl; aminoalkylamino; polyalkylamino orsubstituted silyl, as, among others, described, e.g., in PCT patentapplication WO 99/54459, European patents EP 0 586 520 B1 or EP 0 618925 B1, incorporated by reference in their entireties. As used herein,“alkyl” means C1-C12-alkyl and “lower alkyl” means C1-C6-alkyl,including C1-, C2-, C3-, C4-, C5- and C6-alkyl.

In another aspect, the 5′-end of the antisense strand, the 5′-end of thesense strand, the 3′-end of the antisense strand or the 3′-end of thesense strand is covalently connected to a prodrug moiety. In oneembodiment, the moiety is cleaved in an endosome. In another the moietyis cleaved in the cytoplasm.

Various possible non-limiting embodiments of the siRNA of the presentinvention having different kinds of end modification(s) are presented inthe following Table.

Various embodiments of the interfering ribonucleic acid according to thepresent invention

Various embodiments of the interfering ribonucleic acid according to thepresent invention Antisense strand Sense strand 1.) 5′-end free OH freeOH 3′-end free OH free OH 2.) 5′-end free OH free OH 3′-end endmodification end modification 3.) 5′-end free OH free OH 3′-end free OHend modification 4.) 5′-end free OH free OH 3′-end end modification freeOH 5.) 5′-end free OH end modification 3′-end free OH free OH 6.) 5′-endfree OH end modification 3′-end end modification free OH 7.) 5′-end freeOH end modification 3′-end free OH end modification 8.) 5′-end free OHend modification 3′-end end modification end modification

In another embodiment, the terminal 3′ nucleotide or two terminal3′-nucleotides on either or both of the antisense strand or sense strandis a 2′-deoxynucleotide. In another embodiment, the 2′-deoxynucleotideis a 2′-deoxy-pyrimidine. In another embodiment, the 2′-deoxynucleotideis a 2′ deoxy-thymidine.

shRNA and Linked siRNA:

It is within the present invention that any disclosure related to shRNAand linked siRNA provided herein specifically referring to an antisensestrand or nucleotides forming such antisense strand equally applies toan antisense stretch or nucleotides forming such antisense stretch, andany disclosure related to 5′ and 3′ end modifications modificationprovided herein specifically referring to a sense strand or nucleotidesforming such sense strand equally applies to a sense stretch ornucleotides forming such sense stretch.

In one embodiment, the nucleic acid molecule of the invention and thesiRNA of the invention in particular are shRNA and/or linked siRNA,whereby such shRNA and/or linked siRNA is preferably one describedherein.

It is within the present invention that the double-stranded structure isformed by two separate strands, i.e. the antisense strand and the sensestrand. However, it is also with in the present invention that theantisense strand and the sense strand are covalently linked to eachother.

Such linkage may occur between any of the nucleotides forming theantisense strand and sense strand, respectively. Such linkage can beformed by covalent or non-covalent linkages. Covalent linkage may beformed by linking both strands one or several times and at one orseveral positions, respectively, by a compound preferably selected fromthe group comprising methylene blue and bifunctinoal groups. Suchbifunctional groups are preferably selected from the group comprisingbis(2-chloroethyl)amine, N-acetly-N′-(p-glyoxylbenzoyl)cystamine,4-thiouracile and psoralene.

In one embodiment, the antisense strand and the sense strand are linkedby a loop structure. In another embodiment, of the loop structure iscomprised of a non-nucleic acid polymer. In another embodiment, thenon-nucleic acid polymer is polyethylene glycol. In another embodiment,the 5′-end of the antisense strand is linked to the 3 ‘-terminus of thesense strand. In another embodiment, the 3’-end of the antisense strandis linked to the 5 ′-end of the sense strand.

In another embodiment, the loop consists of a nucleic acid. As usedherein, locked nucleic acid (LNA) (Elayadi and Corey (2001) Curr OpinInvestig Drugs. 2(4):558-61) and peptide nucleic acid (PNA) (reviewed inFaseb J. (2000) 14:1041-1060) are regarded as nucleic acids and may alsobe used as loop forming polymers. In one embodiment, the nucleic acid isribonucleic acid. In one embodiment, the 5′-terminus of the antisensestrand is linked to the 3′-terminus of the sense strand. In anotherembodiment, the 3′-end of the antisense strand is linked to the5′-terminus of the sense strand. The loop consists of a minimum lengthof four nucleotides or nucleotide analogues. In one embodiment, the loopconsists of a length of nucleotides or nucleotide analogues selectedfrom 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. In one embodiment, thelength of the loop is sufficient for linking the two strands covalentlyin a manner that a back folding can occur through a loop structure orsimilar structure. The ribonucleic acid constructs may be incorporatedinto suitable vector systems. Preferably the vector comprises a promoterfor the expression of RNAi. Preferably the respective promoter is polIII and more preferably the promoters are the U6, H1, 7SK promoter asdescribed in Good et al. (1997) Gene Ther. 4, 45-54.

In another embodiment, the nucleic acid according to the presentinvention comprises a phosphorothioate internucleotide linkage. In oneembodiment, a phosphorothioate internucleotide linkage is within 5nucleotides from the 3′-end or the 5′-end of either or both of theantisense strand and the sense strand. The antisense strand can compriseabout one to about five phosphorothioate internucleotide linkages.

Combinations of Embodiments:

It is within the present invention that any disclosure related tocombinations of embodiments provided herein specifically referring to anantisense strand or nucleotides forming such antisense strand equallyapplies to an antisense stretch or nucleotides forming such antisensestretch, and any disclosure related to 5′ and 3′ end modificationsmodification provided herein specifically referring to a sense strand ornucleotides forming such sense strand equally applies to a sense stretchor nucleotides forming such sense stretch.

In one embodiment, an overhang at the 3′-end of the sense strand isselected from consisting of 1, 2, 3, 4 and 5 nucleotides in length. Inone embodiment, an overhang at the 5′-end of the antisense strand isselected from consisting of 1, 2, 3, 4 and 5 nucleotides in length. Inone embodiment, an overhang at the 5′-end of the sense strand isselected from consisting of 1, 2, 3, 4 and 5 nucleotides in length.

In one embodiment, the siRNA molecule is blunt-ended on both ends andhas a length selected from the group consisting of 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28 or 29 consecutive nucleotides.

In one embodiment, the siRNA molecule is blunt-ended on one end and thedouble stranded portion of the siRNA molecule has a length selected fromthe group consisting of 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28 or 29 consecutive nucleotides.

In one embodiment, the siRNA molecule has overhangs on both ends and thedouble stranded portion of the siRNA molecule has a length selected fromthe group consisting of 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28 or 29 consecutive nucleotides.

In one embodiment, the siRNA molecule comprises an overhang, saidoverhang comprising at least one deoxyribonucleotide. In one embodiment,the siRNA molecule comprises an overhang, said overhang comprising twodeoxyribonucleotides.

In one embodiment, the siRNA molecule has overhangs on the 3′-end of theantisense strand and at the 3′-end of the sense strand, said overhangscomprising at least one deoxyribonucleotide. In one embodiment, thesiRNA molecule has overhangs on the 3′-end of the antisense strand andat the 3′-end of the sense strand, said overhangs consisting twodeoxyribonucleotides.

The nucleotide(s) forming the overhang may be (a)desoxyribonucleotide(s), (a) ribonucleotide(s) or a combination thereof.In one embodiment, the antisense strand and/or the sense strand comprisea TT dinucleotide at the 3′ end.

In a preferred embodiment dT as used herein is indicative of adeoxyribonucleotide, namely T, in a molecule which is otherwise an RNAor which consists of ribonucleotides which may, for example, be modifiedas indicated herein.

Processes of Making:

The nucleic acid of the present invention can be produced using routinemethods in the art including chemically synthesis or expressing thenucleic acid either in vitro (e.g., run off transcription) or in vivo.In one embodiment, the siRNA is produced using solid phase chemicalsynthesis. In another embodiment, the nucleic acid is produced using anexpression vector. In one embodiment, the expression vector produced thenucleic acid of the invention in the target cell. Accordingly, suchvector can be used for the manufacture of a medicament. Methods for thesynthesis of the nucleic acid molecule described herein are known to theones skilled in the art. Such methods are, among others, described inCaruthers et al., 1992, Methods in Enzymology 211, 3-19, Thompson etal., International PCT Publication No. WO 99/54459, Wincott et al.,1995, Nucleic Acids Res. 23, 2677-2684, Wincott et al., 1997, MethodsMol. Bio., 74, 59, Brennan et al., 1998, Biotechnol Bioeng., 61, 33-45,and Brennan, U.S. Pat. No. 6,001,311 (each incorporated herein byreference in their entireties).

As used herein in connection with any aspect of the invention a wordingdefining the limits of a range of length such as, e.g., “from 13 to 35”means any integer from 13 to 35, i.e. 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 and 35. In otherwords, any range defined by two integers explicitly mentioned is meantto comprise and disclose any integer defining said limits and anyinteger comprised within said range.

Delivery/Formulations:

siRNA can be delivered to cells, both in vitro and in vivo, by a varietyof methods known to those of skill in the art, including direct contactwith cells (“naked” siRNA) or by in combination with one or more agentsthat facilitate targeting or delivery into cells. Such agents andmethods include nanoemulsions (WO 2009/141257 A1), lipoplexes,liposomes, iontophoresis, hydrogels, cyclodextrins, nanocapsules, micro-and nanospheres and proteinaceous vectors (e.g., Bioconjugate Chem.(1999) 10:1068-1074 and WO 00/53722). The nucleic acid/vehiclecombination may be locally delivered in vivo by direct injection or byuse of an infusion pump. The siRNA of the invention can be delivered invivo by various means including intravenous subcutaneous, intramuscularor intradermal injection or inhalation. The molecules of the instantinvention can be used as pharmaceutical agents. Preferably,pharmaceutical agents prevent, modulate the occurrence, or treat(alleviate a symptom to some extent, preferably all of the symptoms) ofa disease state in a subject. Accordingly, the present invention isrelated in a further aspect to a composition comprising a nucleic acidmolecule of the invention and one of such agents; preferably suchcomposition is for delivery of the nucleic acid molecule of theinvention in any of the methods described herein.

The nanoemulsion as described in international patent application WO2009/141257 A1 is a stable perfluorcarbon nanoemulsion having anendocytosis enhancing surface, whereby the nanoemulsion has adiscontinuous perfluorcarbon phase and a buffered continuous aqueousphase and comprises (a) a perfluorcarbon component comprising at leastone least one perfluorocarbon compound; (b) an emulsifying component;and (c) an endocytosis enhancing component comprising at least onecompound inducing cellular uptake of the nanoemulsion via endocytosis.The nanoemulsion may have a particle size of below 100 nm, preferablythe nanoemulsion consists of particles having an average size of about50 nm. Methods for measuring partice sizes are known in the art and, forexample, described in Murdock R C et al. (R. C. Murdock, et al.,“Characterization of nanomaterial dispersion in solution prior to invitro exposure using dynamic light scattering technique,” Toxicol. Sci.101(2), 239 (2008)) or in Bootz et al. (A. Bootz, V. Vogel, D. Schubert,and J. Kreuter, “Comparison of scanning electron microscopy, dynamiclight scattering and analytical ultracentrifugation for the sizing ofpoly(butyl cyanoacrylate) nanoparticles,” Eur. J. Pharm. Biopharm.57(2), 369 (2004).) Typically, the buffered aqueous phase represents 25to 60 wt. % of the nanoemulsion. In an embodiment, the at least onecompound inducing cellular uptake via endocytosis is selected fromtransferrin, apolipoprotein A1,glycosylphosphatidylinositol(GIP)-anchored proteins, megalinbindingproteins, atennapedia proteins, fragments and derivatives of saidcompounds and compounds having an analogous effect, most preferably saidcompound is transferrin or a fragment or derivative thereof. In anotherembodiment, the at least one perfluorocarbon compound is selected fromC_(m)F_(2m+1X), XC_(m)F_(2m)X, XC_(n)F_(2n)OC_(o)F_(2o)X,N(C_(o)F_(2o)X)₃ and N(C_(o)F_(2o+1))₃ (wherein m is an integer from 3to 10, n and 0 are integers from 1 to 5, and X is independently fromfurther occurrence selected from Cl, Br and I), preferably theperfluorcarbon is selected from perfluoroocytlbromide andperfluorotributylamine and mixtures thereof. The emulsifying componentmay comprise at least one phospholipid as the essential emulsifyingcomponent and one or more helper lipids. The least one phospholipid isselected from compounds represented by the formula I

wherein R¹ and R² are independently selected from H and C₁₆₋₂₄ acylresidues, which may be saturated or unsaturated and may carry 1 to 3residues R³ and wherein one or more of the C-atoms may be substituted by0 or NR⁴, and X is selected from H, —(CH₂)_(p)—N(R⁴)₃ ⁺,—(CH₂)_(p)—CH(N(R⁴)₃ ⁺)—COO—, —(CH₂)_(p)—CH(OH)—CH₂OH and—CH₂(CHOH)_(p)—CH₂OH (wherein p is an integer from 1 to 5; R³ isindependently selected from H, lower alkyl, F, Cl, CN und OH; and R⁴ isindependently selected from H, CH₃ und CH₂CH₃) or a pharmacologicallyacceptable salt thereof, preferably R¹ and R² are independently selectedfrom Hand unsubstituted C₁₆₋₂₄ acyl residues, which may be saturated orunsaturated, and X is selected from a choline, serine, ethanolamine andinositol residue, most preferably the phospholipid component is selectedfrom phosphatidylcholine, Iysophoshatidylcholine,phophatidylethanolamine and mixtures thereof. The helper lipid may beselected from fatty acids, steroids, vitamins and mixtures thereof. In apreferred embodiment the nanoemulsion comprises perfluoroocytlbromide asperfluorcarbon component (a), an emulsifying component (b) comprisingphosphatidylcholine, sphingomyelin, cholesterol andIysophosphatidylcholine, as phospholipid, and transferrin as theendocytosis enhancing component (c).

Lipid nanoparticles comprising phosphatidylcholine which can also beused in the formulation and delivery of the nucleic acid molecule of theinvention are, for example, described in Torchilin V P (V. P. Torchilin,“Recent advances with liposomes as pharmaceutical carriers,” Nat. Rev.Drug Discov. 4(2), 145 (2005)), Ozpolat B et al. (B. Ozpolat, A. K.Sood, and G. Lopez-Berestein, “Nanomedicine based approaches for thedelivery of siRNA in cancer,” J. Intern. Med. 267(1), 44 (2010)) orAbbasalipourkabir R et al. (R. Abbasalipourkabir, A. Salehzadeh, and R.Abdullah, “Characterization and stability of nanostructured lipidcarriers as drug delivery system,” Pak. J. Biol. Sci. 15(3), 141(2012)).

Another means which can be used for the formulation and/or delivery of anucleic acid molecule of the invention are surface-modified liposomescontaining poly (ethylene glycol) lipids (PEG-modified, orlong-circulating liposomes or stealth liposomes). These formulationsoffer a method for increasing stability of a liposome or lipoplexsolutions by preventing their aggregation and fusion. The formulationsalso have the added benefit in vivo of resisting opsonization andelimination by the mononuclear phagocytic system (MPS or RES), therebyenabling longer blood circulation times and enhanced tissue exposure forthe encapsulated drug. Such liposomes have been shown to accumulateselectively in tumors, presumably by extravasation and capture in theneovascularized target tissues (Lasic et al., Science 1995, 267,1275-1276; Oku et al., 1995, Biochim. Biophys. Acta, 1238, 86-90). Thelong-circulating liposomes enhance the pharmacokinetics andpharmacodynamics of DNA and RNA, particularly compared to conventionalcationic liposomes which are known to accumulate in tissues of the MPS(Liu et al., J. Biol. Chem. 1995, 42, 24864-24780; Choi et al.,International PCT Publication No. WO 96/10391; Ansell et al.,International PCT Publication No. WO 96/10390; Holland et al.,International PCT Publication No. WO 96/10392). Long-circulatingliposomes also protect the siRNA from nuclease degradation.

A further means which can be used in the formulation and/or delivery ofa nucleic acid molecule of the invention are lipoplexes as, for example,described in WO 2005/105152. In a preferred embodiment such lipoplex isa positively charged liposome consisting of:

(a) about 50 mol % β-arginyl-2,3-diaminopropionicacid-N-palmityl-N-oleyl-amide trihydrochloride, preferablyβ-(L-arginyl)-2,3-L-diaminopropionic acid-N-palmityl-N-oleyl-amidetri-hydrochloride, (b) about 48 to 49 mol %1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhyPE), and (c) about1 to 2 mol %1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylen-glycole,preferablyN-(Carbonyl-methoxypolyethyleneglycol-2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolaminesodium salt.

Pharmaceutical Compositions

The nucleic acid molecule of the invention may be formulated aspharmaceutical compositions. The pharmaceutical compositions may be usedas medicaments or as diagnostic agents, alone or in combination withother agents. The pharmaceutical compositions may be used in any methodof the invention.

For example, one or more nucleic acid molecules and/or one or moresiRNAs of the invention can be combined with a delivery vehicle (e.g.,nanoemulsion or liposomes) and excipients, such as carriers, diluents.In a preferred embodiment a pharmaceutical composition is a compositionas described in section “delivery/formulations” herein.

Other agents such as preservatives and stabilizers can also be added.Methods for the delivery of nucleic acid molecules are known in the artand described, e.g., in Akhtar et al., 1992, Trends Cell Bio., 2, 139;Delivery Strategies for Antisense Oligonucleotide Therapeutics, ed.Akhtar, 1995, Maurer et al., 1999, Mol. Memb. Biol., 16, 129-140;Hofland and Huang, 1999, Handb. Exp. Pharmacol., 137, 165-192; and Leeet al., 2000, ACS Symp. Ser., 752, 184-192, U.S. Pat. No. 6,395,713 andPCT WO 94/02595 (each of which are incorporated herein by reference intheir entireties). The siRNA of the present invention can also beadministered in combination with other therapeutic compounds, eitheradministrated separately or simultaneously, e.g., as a combined unitdose. In one embodiment, the invention includes a pharmaceuticalcomposition comprising one or more siRNA according to the presentinvention in a physiologically/pharmaceutically acceptable excipient,such as a stabilizer, preservative, diluent, buffer, and the like.

Dosage levels for the medicament and pharmaceutical compositions of theinvention can be determined by those skilled in the art by routineexperimentation. In one embodiment, a unit dose contains between about0.01 mg/kg and about 100 mg/kg body weight of siRNA. In one embodiment,the dose of siRNA is about 10 mg/kg and about 25 mg/kg body weight. Inone embodiment, the dose of siRNA is about 1 mg/kg and about 10 mg/kgbody weight. In one embodiment, the dose of siRNA is about 0.05 mg/kgand about 5 mg/kg body weight. In another embodiment, the dose of siRNAis about 0.1 mg/kg and about 5 mg/kg body weight. In another embodiment,the dose of siRNA is about 0.1 mg/kg and about 1 mg/kg body weight. Inanother embodiment, the dose of siRNA is about 0.1 mg/kg and about 0.5mg/kg body weight. In another embodiment, the dose of siRNA is about 0.5mg/kg and about 1 mg/kg body weight.

In one aspect, the pharmaceutical composition is a sterile injectableaqueous suspension or solution. In one aspect, the pharmaceuticalcomposition is in lyophilized form. In one embodiment the pharmaceuticalcomposition comprises a nanoemulsion comprising a siRNA molecule of theinvention. In one embodiment, the pharmaceutical composition compriseslyophilized lipoplexes, wherein the lipoplexes comprises a siRNA of thepresent invention. In another embodiment, the pharmaceutical compositioncomprises an aqueous suspension of lipoplexes, wherein the lipoplexescomprises a siRNA of the present invention.

The pharmaceutical compositions and medicaments of the present inventionmay be administered to a subject (mammal) in the disclosed methods oftreatment. In one embodiment, the mammal is selected from the groupconsisting humans, dogs, cats, horses, cattle, pig, goat, sheep, mouse,rat, hamster and guinea pig. In one embodiment, the mammal is a human.In another embodiment, the mammal is a non-human mammal.

Kits

In a further aspect the invention is related to a kit. The kit comprisesa nucleic acid molecule of the invention, preferably a siRNA of theinvention, and at least one selected from the group of a container, aninstruction leaflet, a buffer, a positive control, a negative control, adelivery agent or delivery agent, whereby the delivery agent ispreferably one disclosed herein, and a reaction mixture. The kit isuseful in/suitable for the practicing of any method of the invention. Inan embodiment the kit is for use in any method of the invention.

Methods of Treatment

The nucleic acid molecule of the invention is useful in and may be usedin the treatment and/or prevention of a disease. In an embodiment, themethod comprises the administration of a nucleic acid to a subject.Preferably, the subject is suffering from the disease or at risk ofsuffering from the disease. Preferably, the subject is a mammal. Aspreferably used herein, a mammal is an animal selected from the groupcomprising man, ape, monkey, mouse, rat rabbit, cat, dog, cattle, horse,domestic animal, working animal and companion animal. More preferably,the subject is man.

The nucleic acid molecule of the invention is administered to thesubject at an effective amount. Preferably such effective amount is apharmaceutically effective amount or a therapeutically effective amount.

The nucleic acid molecule of the invention can be administered, inprinciple, in any form known to the ones skilled in the art. A preferredroute of administration is systemic administration, more preferably byparenteral administration, preferably by injection. Alternatively, themedicament may be administered locally. Other routes of administrationcomprise intramuscular, intraperitoneal, and subcutaneous, per orum,intranasal, intratracheal or pulmonary with preference given to theroute of administration that is the least invasive, while ensuringefficacy.

Parenteral administration is generally used for subcutaneous,intramuscular or intravenous injections and infusions. Additionally, oneapproach for parenteral administration employs the implantation of aslow-release or sustained-released systems, which assures that aconstant level of dosage is maintained, that are well known to theordinary skill in the art.

Furthermore, preferred medicaments of the present invention can beadministered in intranasal form via topical use of suitable intranasalvehicles, inhalants, or via transdermal routes, using those forms oftransdermal skin patches well known to those of ordinary skill in thatart. To be administered in the form of a transdermal delivery system,the dosage administration will, of course, be continuous rather thanintermittent throughout the dosage regimen. Other preferred topicalpreparations include creams, ointments, lotions, aerosol sprays andgels.

In a preferred embodiment the nucleic acid molecule of the invention isa siRNA of the invention.

In an embodiment, the method for the treatment and/or prevention of adisease is a disease which can be treated by decreasing the expressionof the Notch 1 gene and more specifically by decreasing the translationof the mRNA coding for Notch 1. Specific diseases and medical conditionsfalling within this group of diseases are known to a person skilled inthe art. Furthermore, methods for determining this kind of disease areequally known to a person skilled in the art. A preferred disease iscancer, preferably those forms of cancer where Notch 1 is up-regulated,Notch 1 is expressed in an altered manner compared to non-disease tissueor non-diseased tissue and/or where a therapeutic effect may be achievedby decreasing the expression of the Notch 1 gene and more specificallyby decreasing the translation of the mRNA coding for Notch 1.

In an embodiment the disease is one of the following group, where theinvolvement of Notch 1 has been demonstrated and, respectively, Notch 1been identified as a drugable target: Esophageal cancer (see, Streppel,E. A. Montgomery, and A. Maitra, “New Advances in the Pathogenesis andProgression of Barrett's Esophagus,” Curr. Mol. Med. (2013)), oralsquamous cell carcinoma (see, R. Yoshida, et al., “The pathologicalsignificance of Notch1 in oral squamous cell carcinoma,” Lab Invest(2013)), head and neck cancer (see, J. T. Lin, et al., “Association ofhigh levels of Jagged-1 and Notch-1 expression with poor prognosis inhead and neck cancer,” Ann. Surg. Oncol. 17(11), 2976 (2010)), tonguecancer (see Y. H. Joo, C. K. Jung, M. S. Kim, and D. I. Sun,“Relationship between vascular endothelial growth factor and Notch1expression and lymphatic metastasis in tongue cancer,” Otolaryngol. HeadNeck Surg. 140(4), 512 (2009)), leukemia (see E. Kanamori, et al., “Flowcytometric analysis of Notch1 and Jagged1 expression in normal bloodcells and leukemia cells,” Exp. Ther. Med. 4(3), 397 (2012); and Zhang Jet al. (J. Zhang, et al., “Prognostic impact of delta-like ligand 4 andNotch1 in acute myeloid leukemia,” Oncol. Rep. 28(4), 1503 (2012)),renal cell carcinoma (see, Q. Ai, et al., “High-level expression ofNotch1 increased the risk of metastasis in T1 stage clear cell renalcell carcinoma,” PLoS. One. 7(4), e35022 (2012) and J. Sjolund, et al.,“The notch and TGF-beta signaling pathways contribute to theaggressiveness of clear cell renal cell carcinoma,” PLoS. One. 6(8),e23057 (2011)), gastric cancer (see, T. S. Yeh, et al., “The activatedNotch1 signal pathway is associated with gastric cancer progressionthrough cyclooxygenase-2,” Cancer Res. 69(12), 5039 (2009), and Y. Sun,et al., “Differential Notch1 and Notch2 expression and frequentactivation of Notch signaling in gastric cancers,” Arch. Pathol. LabMed. 135(4), 451 (2011)), colon adenocarcinoma (see, M. Reedijk, et al.,“Activation of Notch signaling in human colon adenocarcinoma,” Int J.Oncol. 33(6), 1223 (2008) and M. Reedijk, et al., “Activation of Notchsignaling in human colon adenocarcinoma,” Int J. Oncol. 33(6), 1223(2008)), endometrial cancer/uterine corpus (see Y. Mitsuhashi, et al.,“Prognostic significance of Notch signalling molecules and theirinvolvement in the invasiveness of endometrial carcinoma cells,”Histopathology 60(5), 826 (2012)), cervical cancer/uterine cervix (see,L. Santos, et al., “Identification of differential expressed transcriptsin cervical cancer of Mexican patients,” Tumour. Biol. 32(3), 561(2011)), intrahepatic cholangiocarcinoma (see, Q. Zhou, et al., “Theroles of Notch1 expression in the migration of intrahepaticcholangiocarcinoma,” BMC. Cancer 13, 244 (2013), and S. Zender, et al.,“A critical role for notch signaling in the formation ofcholangiocellular carcinomas,” Cancer Cell 23(6), 784 (2013)),hepatocellular carcinoma (see, A. Villanueva, et al., “Notch signalingis activated in human hepatocellular carcinoma and induces tumorformation in mice,” Gastroenterology 143(6), 1660 (2012), and R. Fan, etal., “Cooperation of deregulated Notch signaling and Ras pathway inhuman hepatocarcinogenesis,” J. Mol. Histol. 42(5), 473 (2011)),osteosarcoma (see, J. Yang and W. Zhang, “New molecular insights intoosteosarcoma targeted therapy,” Curr. Opin. Oncol. 25(4), 398 (2013)),urinary bladder carcinoma (see, A. G. Abdou, et al.,“Immunohistochemical analysis of the role and relationship betweenNotch-1 and Oct-4 expression in urinary bladder carcinoma,” APMIS(2013)), malignant melanoma (see, C. S. Muller, “Notch signaling andmalignant melanoma,” Adv. Exp. Med. Biol. 727, 258 (2012)), thyroidcancer (see, H. S. Park, et al., “Notch1 receptor as a marker of lymphnode metastases in papillary thyroid cancer,” Cancer Sci. 103(2), 305(2012)), lung adenocarcinoma (see K. A. Hassan, et al., “Notch pathwayactivity identifies cells with cancer stem cell-like properties andcorrelates with worse survival in lung adenocarcinoma,” Clin. CancerRes. 19(8), 1972 (2013), and B. Westhoff, et al., “Alterations of theNotch pathway in lung cancer,” Proc. Natl. Acad. Sci. U. S. A 106(52),22293 (2009)), prostata cancer (see, H. Zhu, et al., “Elevated Jagged-1and Notch-1 expression in high grade and metastatic prostate cancers,”Am. J. Transl. Res. 5(3), 368 (2013)) and M. Kashat, et al.,“Inactivation of AR and Notch-1 signaling by miR-34a attenuates prostatecancer aggressiveness,” Am. J. Transl. Res. 4(4), 432 (2012)), breastcancer (see, J. Speiser, et al., “Notch-1 and Notch-4 biomarkerexpression in triple-negative breast cancer,” Int J. Surg. Pathol.20(2), 139 (2012), and S. Mittal, et al., “Cooperation of Notch andRas/MAPK signaling pathways in human breast carcinogenesis,” Mol. Cancer8, 128 (2009)), ovarian cancer (see, S. L. Rose, M. Kunnimalaiyaan, J.Drenzek, and N. Seiler, “Notch 1 signaling is active in ovarian cancer,”Gynecol. Oncol. 117(1), 130 (2010)), pancreatic cancer (see, F. H.Sarkar, S. Banerjee, and Y. Li, “Pancreatic cancer: pathogenesis,prevention and treatment,” Toxicol. Appl. Pharmacol. 224(3), 326 (2007),0. JP De La, et al., “Notch and Kras reprogram pancreatic acinar cellsto ductal intraepithelial neoplasia,” Proc. Natl. Acad. Sci. U. S. A105(48), 18907 (2008), E. Ristorcelli and D. Lombardo, “Targeting Notchsignaling in pancreatic cancer,” Expert. Opin. Ther. Targets. 14(5), 541(2010), Z. Wang, et al., “Notch-1 down-regulation by curcumin isassociated with the inhibition of cell growth and the induction ofapoptosis in pancreatic cancer cells,” Cancer 106(11), 2503 (2006), P.Buehler, et al., “The Notch signaling pathway is related toneurovascular progression of pancreatic cancer,” Ann. Surg. 242(6), 791,discussion (2005), Z. Wang, et al., “Down-regulation of Notch-1contributes to cell growth inhibition and apoptosis in pancreatic cancercells,” Mol. Cancer Ther. 5(3), 483 (2006), Z. Wang, et al.,“Down-regulation of notch-1 inhibits invasion by inactivation of nuclearfactor-kappaB, vascular endothelial growth factor, and matrixmetalloproteinase-9 in pancreatic cancer cells,” Cancer Res. 66(5), 2778(2006)), and glioma (see, X. Zhang, et al., “Notch1 promotes glioma cellmigration and invasion by stimulating beta-catenin and NF-kappaBsignaling via AKT activation,” Cancer Sci. 103(2), 181 (2012), L. Jiang,et al., “Notch1 expression is upregulated in glioma and is associatedwith tumor progression,” J. Clin. Neurosci. 18(3), 387 (2011), J. Li, etal., “Notch1 is an independent prognostic factor for patients withglioma,” J. Surg. Oncol. 103(8), 813 (2011), and S. Puget, et al.,“Candidate genes on chromosome 9q33-34 involved in the progression ofchildhood ependymomas,” J. Clin. Oncol. 27(11), 1884 (2009)).

It is within the present invention that in addition to a nucleic acidmolecule of the invention at least one further therapeutically orpharmaceutically active agent (also referred to herein as “second offurther agent”) is used in the methods of treatment. Such method oftreatment is also referred to as combination therapy.

“Combination therapy” (or “co-therapy”) includes the administration of anucleic acid molecule of the invention and at least a second or furtheragent as part of a specific treatment regimen intended to provide thebeneficial effect from the co-action of these therapeutic agents, i.e.the medicament of the present invention and said second or furtheragent. The beneficial effect of the combination includes, but is notlimited to, pharmacokinetic or pharmacodynamic co-action resulting fromthe combination of therapeutic agents. Administration of thesetherapeutic agents in combination typically is carried out over adefined time period (usually minutes, hours, days or weeks dependingupon the combination selected).

“Combination therapy” may be, but generally is not, intended toencompass the administration of two or more of these therapeutic agentsas part of separate monotherapy regimens.

“Combination therapy” is intended to embrace administration of thesetherapeutic agents in a sequential manner, that is, wherein eachtherapeutic agent is administered at a different time, as well asadministration of these therapeutic agents, or at least two of thetherapeutic agents, in a substantially simultaneous manner.Substantially simultaneous administration can be accomplished, forexample, by administering to a subject a single capsule having a fixedratio of each therapeutic agent or in multiple, single capsules for eachof the therapeutic agents.

Sequential or substantially simultaneous administration of eachtherapeutic agent can be effected by any appropriate route including,but not limited to, topical routes, oral routes, intravenous routes,intramuscular routes, and direct absorption through mucous membranetissues. The therapeutic agents can be administered by the same route orby different routes. For example, a first therapeutic agent of thecombination selected may be administered by injection while the othertherapeutic agents of the combination may be administered topically.Alternatively, for example, all therapeutic agents may be administeredtopically or all therapeutic agents may be administered by injection.The sequence in which the therapeutic agents are administered is notnarrowly critical unless noted otherwise. “Combination therapy” also canembrace the administration of the therapeutic agents as described abovein further combination with other biologically active ingredients. Wherethe combination therapy further comprises a non-drug treatment, thenon-drug treatment may be conducted at any suitable time so long as abeneficial effect from the co-action of the combination of thetherapeutic agents and non-drug treatment is achieved. For example, inappropriate cases, the beneficial effect is still achieved when thenon-drug treatment is temporally removed from the administration of thetherapeutic agents, perhaps by days or even weeks.

In accordance therewith such further therapeutically or pharmaceuticallyactive agent is also administered to the subject. In an embodiment, thefurther therapeutically or pharmaceutically active agent is administeredprior, together with or after the nucleic acid molecule of theinvention. In an embodiment the further therapeutically orpharmaceutically active agent is one selected from the group comprisingtaxane derivates such as docetaxel, paclitaxel (see, Q. F. Ye, et al.,“siRNA-mediated silencing of Notch-1 enhances docetaxel induced mitoticarrest and apoptosis in prostate cancer cells,” Asian Pac. J. CancerPrev. 13(6), 2485 (2012), C. C. Zhang, et al., “Synergistic effect ofthe gamma-secretase inhibitor PF-03084014 and docetaxel in breast cancermodels,” Stem Cells Transl. Med. 2(3), 233 (2013), K. A. Hassan, et al.,“Notch pathway activity identifies cells with cancer stem cell-likeproperties and correlates with worse survival in lung adenocarcinoma,”Clin. Cancer Res. 19(8), 1972 (2013), Y. P. Liu, et al., “Cisplatinselects for multidrug-resistant CD133+ cells in lung adenocarcinoma byactivating Notch signaling,” Cancer Res. 73(1), 406 (2013), and S. Zang,et al., “RNAi-mediated knockdown of Notch-1 leads to cell growthinhibition and enhanced chemosensitivity in human breast cancer,” Oncol.Rep. 23(4), 893 (2010)), platinum derivatives such as cisplatin andoxaliplatin (see, K. A. Hassan, et al., “Notch pathway activityidentifies cells with cancer stem cell-like properties and correlateswith worse survival in lung adenocarcinoma,” Clin. Cancer Res. 19(8),1972 (2013) and Z. P. Zhang, et al., “Correlation of Notch1 expressionand activation to cisplatin-sensitivity of head and neck squamous cellcarcinoma,” Ai. Zheng. 28(2), 100 (2009)), Nucleoside analogues such as5-fluorouracil (see, R. D. Meng, et al., “gamma-Secretase inhibitorsabrogate oxaliplatin-induced activation of the Notch-1 signaling pathwayin colon cancer cells resulting in enhanced chemosensitivity,” CancerRes. 69(2), 573 (2009)), topoisomerase I inhibitors such as irinotecan(see, R. D. Meng, et al., “gamma-Secretase inhibitors abrogateoxaliplatin-induced activation of the Notch-1 signaling pathway in coloncancer cells resulting in enhanced chemosensitivity,” Cancer Res. 69(2),573 (2009), intercalating substances such as doxorubicin (see, Y. P.Liu, et al., “Cisplatin selects for multidrug-resistant CD133+ cells inlung adenocarcinoma by activating Notch signaling,” Cancer Res. 73(1),406 (2013)), nucleoside analogues such as gemcitabine (see, X. Du, etal., “Notch1 contributes to chemoresistance to gemcitabine and serves asan unfavorable prognostic indicator in pancreatic cancer,” World J.Surg. 37(7), 1688 (2013), S. Yabuuchi, et al., “Notch signaling pathwaytargeted therapy suppresses tumor progression and metastatic spread inpancreatic cancer,” Cancer Lett. 335(1), 41 (2013), and S. Richter, etal., “A phase I study of the oral gamma secretase inhibitor R04929097 incombination with gemcitabine in patients with advanced solid tumors(PHL-078/CTEP 8575),” Invest New Drugs (2013)), syntheticglucocorticoids such as dexamethasone (see, Q. Zhou, et al., “The rolesof Notch1 expression in the migration of intrahepaticcholangiocarcinoma,” BMC. Cancer 13, 244 (2013)) and alkylating agentssuch as temozolomide (see, C. A. Gilbert, M. C. Daou, R. P. Moser, andA. H. Ross, “Gamma-secretase inhibitors enhance temozolomide treatmentof human gliomas by inhibiting neurosphere repopulation and xenograftrecurrence,” Cancer Res. 70(17), 6870 (2010)).

An embodiment of the method of the invention whereby the method oftreatment is a combination therapy is one where rather thanadministering at least one further pharmaceutically or therapeuticallyactive agent the subject receives radiotherapy.

Radiotherapy (also referred to X-ray therapy or irradiation) is the useof ionizing radiation to kill cancer cells. Radiotherapy is used in themedical art to treat almost every type of solid tumor. Irradiation isalso used to treat leukemia and lymphoma. Radiotherapy injures ordestroys cells in the area being treated by damaging their geneticmaterial, making it impossible for these cells to continue to grow anddivide. The effects of radiotherapy are localized and confined to theregion being treated. Radiation dose to each site depends on a number offactors, including the radiosensitivity of each cancer type and whetherthere are tissues and organs nearby that may be damaged by radiation.The goal of radiotherapy is to damage as many cancer cells as possible,while limiting harm to nearby healthy tissue.

In a further embodiment of the method of the invention for the treatmentand/or prevention of a disease comprising the administration of anucleic acid molecule of the invention, preferably a siRNA of theinvention, to a subject, whereby the disease is preferably cancer andmore preferably a cancer as disclosed herein, the method is actually anadjunct therapy of adjunctive therapy. The purpose of such adjuncttherapy is to assist a primary treatment, preferably a primary cancertreatment.

The nucleic acid molecule of the invention is useful in and may be usedin a method for restoring drug sensitivity of cancer cells. In anembodiment, the method comprises the administration of a nucleic acid ofthe invention to a subject, whereby the subject is suffering from adisease, preferably cancer, and cancer cells which are involved in thedisease and/or cells which are to be addressed, damaged and/or destroyedby any therapy supplied to the subject or by any pharmaceutically ortherapeutically active agent administered to the subject in thetreatment of the disease, are not or no longer susceptible to suchtherapy and/or such pharmaceutically or therapeutically active agent.Typically, after administration of the nucleic acid molecule of theinvention, said cells become susceptible to such therapy and/orpharmaceutically or therapeutically active agent again, at least to atherapeutically and/or pharmaceutically relevant level. Such therapy ispreferably cancer therapy including, but not limited to, cytostaticbased therapy and radiation therapy, and such pharmaceutically ortherapeutically active agent is one used in cancer therapy. Preferably,the nucleic acid molecule of the invention is a siRNA of the invention.

Insofar, the method for restoring drug sensitivity of cancer cells is amethod for re-sensitizing cancer cells which are not or no longersusceptible to cancer therapy and/or pharmaceutically or therapeuticallyactive agent used in cancer therapy. It will also be acknowledged thatthe method for restoring drug sensitivity of cancer is an adjuncttherapy for a method for the treatment of cancer.

It will be acknowledged that what is disclosed herein in connection withthe method for the treatment and/or prevention of a disease is equallyapplicable to the method for restoring drug sensitivity of cancer cells.This applies in particular to the aspects of such method related to thesubject of the method, the administration and administration routes ofthe nucleic acid of the invention and the like. Insofar, the method forrestoring drug sensitivity of cancer cells is an embodiment of themethod for the treatment and/or prevention of a disease. Preferred formsof cancer which may establish a resistance to a therapy typicallyapplied to a subject suffering from such forms of cancer, are thefollowings:

Esophageal cancer (see, Streppel, E. A. Montgomery, and A. Maitra, “NewAdvances in the Pathogenesis and Progression of Barrett's Esophagus,”Curr. Mol. Med. (2013)), oral squamous cell carcinoma (see, R. Yoshida,et al., “The pathological significance of Notch1 in oral squamous cellcarcinoma,” Lab Invest (2013)), head and neck cancer (see, J. T. Lin, etal., “Association of high levels of Jagged-1 and Notch-1 expression withpoor prognosis in head and neck cancer,” Ann. Surg. Oncol. 17(11), 2976(2010)), tongue cancer (see Y. H. Joo, C. K. Jung, M. S. Kim, and D. I.Sun, “Relationship between vascular endothelial growth factor and Notch1expression and lymphatic metastasis in tongue cancer,” Otolaryngol. HeadNeck Surg. 140(4), 512 (2009)), leukemia (see E. Kanamori, et al., “Flowcytometric analysis of Notch1 and Jagged1 expression in normal bloodcells and leukemia cells,” Exp. Ther. Med. 4(3), 397 (2012); and Zhang Jet al. (J. Zhang, et al., “Prognostic impact of delta-like ligand 4 andNotch1 in acute myeloid leukemia,” Oncol. Rep. 28(4), 1503 (2012)),renal cell carcinoma (see, Q. Ai, et al., “High-level expression ofNotch1 increased the risk of metastasis in T1 stage clear cell renalcell carcinoma,” PLoS. One. 7(4), e35022 (2012) and J. Sjolund, et al.,“The notch and TGF-beta signaling pathways contribute to theaggressiveness of clear cell renal cell carcinoma,” PLoS. One. 6(8),e23057 (2011)), gastric cancer (see, T. S. Yeh, et al., “The activatedNotch1 signal pathway is associated with gastric cancer progressionthrough cyclooxygenase-2,” Cancer Res. 69(12), 5039 (2009), and Y. Sun,et al., “Differential Notch1 and Notch2 expression and frequentactivation of Notch signaling in gastric cancers,” Arch. Pathol. LabMed. 135(4), 451 (2011)), colon adenocarcinoma (see, M. Reedijk, et al.,“Activation of Notch signaling in human colon adenocarcinoma,” Int J.Oncol. 33(6), 1223 (2008) and M. Reedijk, et al., “Activation of Notchsignaling in human colon adenocarcinoma,” Int J. Oncol. 33(6), 1223(2008)), endometrial cancer/uterine corpus (see Y. Mitsuhashi, et al.,“Prognostic significance of Notch signalling molecules and theirinvolvement in the invasiveness of endometrial carcinoma cells,”Histopathology 60(5), 826 (2012)), cervical cancer/uterine cervix (see,L. Santos, et al., “Identification of differential expressed transcriptsin cervical cancer of Mexican patients,” Tumour. Biol. 32(3), 561(2011)), intrahepatic cholangiocarcinoma (see, Q. Zhou, et al., “Theroles of Notch1 expression in the migration of intrahepaticcholangiocarcinoma,” BMC. Cancer 13, 244 (2013), and S. Zender, et al.,“A critical role for notch signaling in the formation ofcholangiocellular carcinomas,” Cancer Cell 23(6), 784 (2013)),hepatocellular carcinoma (see, A. Villanueva, et al., “Notch signalingis activated in human hepatocellular carcinoma and induces tumorformation in mice,” Gastroenterology 143(6), 1660 (2012), and R. Fan, etal., “Cooperation of deregulated Notch signaling and Ras pathway inhuman hepatocarcinogenesis,” J. Mol. Histol. 42(5), 473 (2011)),osteosarcoma (see, J. Yang and W. Zhang, “New molecular insights intoosteosarcoma targeted therapy,” Curr. Opin. Oncol. 25(4), 398 (2013)),urinary bladder carcinoma (see, A. G. Abdou, et al.,“Immunohistochemical analysis of the role and relationship betweenNotch-1 and Oct-4 expression in urinary bladder carcinoma,” APMIS(2013)), malignant melanoma (see, C. S. Muller, “Notch signaling andmalignant melanoma,” Adv. Exp. Med. Biol. 727, 258 (2012)), thyroidcancer (see, H. S. Park, et al., “Notch1 receptor as a marker of lymphnode metastases in papillary thyroid cancer,” Cancer Sci. 103(2), 305(2012)), lung adenocarcinoma (see K. A. Hassan, et al., “Notch pathwayactivity identifies cells with cancer stem cell-like properties andcorrelates with worse survival in lung adenocarcinoma,” Clin. CancerRes. 19(8), 1972 (2013), and B. Westhoff, et al., “Alterations of theNotch pathway in lung cancer,” Proc. Natl. Acad. Sci. U. S. A 106(52),22293 (2009)), prostata cancer (see, H. Zhu, et al., “Elevated Jagged-1and Notch-1 expression in high grade and metastatic prostate cancers,”Am. J. Transl. Res. 5(3), 368 (2013)) and M. Kashat, et al.,“Inactivation of AR and Notch-1 signaling by miR-34a attenuates prostatecancer aggressiveness,” Am. J. Transl. Res. 4(4), 432 (2012)), breastcancer (see, J. Speiser, et al., “Notch-1 and Notch-4 biomarkerexpression in triple-negative breast cancer,” Int J. Surg. Pathol.20(2), 139 (2012), and S. Mittal, et al., “Cooperation of Notch andRas/MAPK signaling pathways in human breast carcinogenesis,” Mol. Cancer8, 128 (2009)), ovarian cancer (see, S. L. Rose, M. Kunnimalaiyaan, J.Drenzek, and N. Seiler, “Notch 1 signaling is active in ovarian cancer,”Gynecol. Oncol. 117(1), 130 (2010)), pancreatic cancer (see, F. H.Sarkar, S. Banerjee, and Y. Li, “Pancreatic cancer: pathogenesis,prevention and treatment,” Toxicol. Appl. Pharmacol. 224(3), 326 (2007),0. JP De La, et al., “Notch and Kras reprogram pancreatic acinar cellsto ductal intraepithelial neoplasia,” Proc. Natl. Acad. Sci. U. S. A105(48), 18907 (2008), E. Ristorcelli and D. Lombardo, “Targeting Notchsignaling in pancreatic cancer,” Expert. Opin. Ther. Targets. 14(5), 541(2010), Z. Wang, et al., “Notch-1 down-regulation by curcumin isassociated with the inhibition of cell growth and the induction ofapoptosis in pancreatic cancer cells,” Cancer 106(11), 2503 (2006), P.Buchler, et al., “The Notch signaling pathway is related toneurovascular progression of pancreatic cancer,” Ann. Surg. 242(6), 791,discussion (2005), Z. Wang, et al., “Down-regulation of Notch-1contributes to cell growth inhibition and apoptosis in pancreatic cancercells,” Mol. Cancer Ther. 5(3), 483 (2006), Z. Wang, et al.,“Down-regulation of notch-1 inhibits invasion by inactivation of nuclearfactor-kappaB, vascular endothelial growth factor, and matrixmetalloproteinase-9 in pancreatic cancer cells,” Cancer Res. 66(5), 2778(2006)), and glioma (see, X. Zhang, et al., “Notch1 promotes glioma cellmigration and invasion by stimulating beta-catenin and NF-kappaBsignaling via AKT activation,” Cancer Sci. 103(2), 181 (2012), L. Jiang,et al., “Notch1 expression is upregulated in glioma and is associatedwith tumor progression,” J. Clin. Neurosci. 18(3), 387 (2011), J. Li, etal., “Notch1 is an independent prognostic factor for patients withglioma,” J. Surg. Oncol. 103(8), 813 (2011), and S. Puget, et al.,“Candidate genes on chromosome 9q33-34 involved in the progression ofchildhood ependymomas,” J. Clin. Oncol. 27(11), 1884 (2009)).

Resistance of cancer cells which can be overcome by the method forrestoring drug sensitivity is Notch 1-induced resistance and Notch1-induced chemoresistance in particular. Insofar, the method forrestoring drug sensitivity of cancer cells is a method for reversingNotch 1-induced resistance and Notch 1-induced chemoresistance inparticular. Whether a cell and a cancer cell in particular is resistantto chemotherapeutics may be determined by routine tests known to aperson skilled in the art such as the MMT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)-assay andflow cytometry.

The MMT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide)-assay measures the reduction of MTT by cellular enzymes. Bymeasuring this metabolic activity via NAD(P)H-dependent enzymes it ispossible to estimate the number of viable cells. Tetrazolium dye assaysallow measurements of both cytotoxicity and cytostatic activity ofchemotherapeutic agents.

In flow cytometry cells are suspended in a fluid-stream and pass througha detector. Using Laser-technology, it allows to determine the number ofcells and to identify biomarkers. A large number of particles can besimultaneous analyzed for biophysical and chemical parameters. Usingthis technique, it is possible to discriminate viable from apoptoticcells to measure the effects of pharmaceutical agents.

Notch 1 induced chemoresistance is, for example, described in K. M.Capaccione and S. R. Pine (K. M. Capaccione and S. R. Pine, “The Notchsignaling pathway as a mediator of tumor survival,” Carcinogenesis34(7), 1420 (2013)). From this and other references it is plausible thatby inhibition the expression of Notch 1 and thus by using the nucleicacid molecule of the invention Notch 1 induced chemoresistance can beovercome. Such other references include, but are not limited to Ye, Q Fet al. (Q. F. Ye, et al., “siRNA-mediated silencing of Notch-1 enhancesdocetaxel induced mitotic arrest and apoptosis in prostate cancercells,” Asian Pac. J. Cancer Prev. 13(6), 2485 (2012)) for prostatecancer, Zhang C C et al. (C. C. Zhang, et al., “Synergistic effect ofthe gamma-secretase inhibitor PF-03084014 and docetaxel in breast cancermodels,” Stem Cells TransL Med. 2(3), 233 (2013)) or Zang S et al. (S.Zang, et al., “RNAi-mediated knockdown of Notch-1 leads to cell growthinhibition and enhanced chemosensitivity in human breast cancer,” Oncol.Rep. 23(4), 893 (2010)) for breast cancer, Hassan K A et al. (K. A.Hassan, et al., “Notch pathway activity identifies cells with cancerstem cell-like properties and correlates with worse survival in lungadenocarcinoma,” Clin. Cancer Res. 19(8), 1972 (2013)) for lung cancer,Zhang Z P et al. (Z. P. Zhang, et al., “Correlation of Notch1 expressionand activation to cisplatin-sensitivity of head and neck squamous cellcarcinoma,” Ai. Zheng. 28(2), 100 (2009)) for squamous cell carcinomas,Meng R D et al. (R. D. Meng, et al., “gamma-Secretase inhibitorsabrogate oxaliplatin-induced activation of the Notch-1 signaling pathwayin colon cancer cells resulting in enhanced chemosensitivity,” CancerRes. 69(2), 573 (2009)) for colon cancer, Liu Y P et al. (Y. P. Liu, etal., “Cisplatin selects for multidrug-resistant CD133+ cells in lungadenocarcinoma by activating Notch signaling,” Cancer Res. 73(1), 406(2013)) for non-small lung cancer, Du X et al. (X. Du, et al., “Notch1contributes to chemoresistance to gemcitabine and serves as anunfavorable prognostic indicator in pancreatic cancer,” World J. Surg.37(7), 1688 (2013)) or Yabuuchi S et al (S. Yabuuchi, et al., “Notchsignaling pathway targeted therapy suppresses tumor progression andmetastatic spread in pancreatic cancer,” Cancer Lett. 335(1), 41 (2013))for pancreatic cancer, Zhou Q et al. (Q. Zhou, et al., “The roles ofNotch1 expression in the migration of intrahepatic cholangiocarcinoma,”BMC. Cancer 13, 244 (2013)) for leukemia and T-cell acute lymphoblasticleukemia in particular, and Gilbert C A et al. (C. A. Gilbert, M. C.Daou, R. P. Moser, and A. H. Ross, “Gamma-secretase inhibitors enhancetemozolomide treatment of human gliomas by inhibiting neurosphererepopulation and xenograft recurrence,” Cancer Res. 70(17), 6870 (2010))for glioma.

Apart from overcoming Notch 1 induced resistance, the nucleic acidmolecule of the invention is also suitable to overcome resistance toradiation as evident from Hovinga K E (K. E. Hovinga, et al.,“Inhibition of notch signaling in glioblastoma targets cancer stem cellsvia an endothelial cell intermediate,” Stem Cells 28(6), 1019 (2010))and Wang J et al. (J. Wang, et al., “Notch promotes radioresistance ofglioma stem cells,” Stem Cells 28(1), 17 (2010)).

The present invention is further illustrated by the figures, examplesand the sequence listing from which further features, embodiments andadvantages may be taken, wherein

FIG. 1 is a diagram indicating the relative expression of Notch 1 upontransfection of C4-2 cells using various siRNAs;

FIG. 2 is a diagram for various siRNAs indicating the remaining relativeNotch 1 mRNA expression as a function of the concentration of theindividual siRNA allowing the determination of the 1050 value for eachsiRNA;

FIGS. 3A and 3B are diagrams showing relative Notch 1 mRNA expressionupon exposure of C4-2 cells to siRNA XD-00404 (FIG. 3A) or XD-00409(FIG. 3B) in its unmodified, intermediate or fully modified form at aconcentration of 10 nM, 1 nm and 0.1 nM;

FIG. 4 is a diagram showing the relative increase in tumour volume(indicated as % SEM) in a xenograft mouse model using PANC-1 cells; and

FIG. 5 is a diagram showing the number of animals in a PANC-1 orthotopictumor model without metastases, with several metastases and with singlemetastases upon treatment of the animal model with control, gemcitabineor a combination of gemcitabine and nanocarrier comprising the siRNA ofthe invention.

EXAMPLE 1 Materials and Methods

siRNAs

The siRNAs represented in Table 1 were prepared using standard chemicalsynthesis:

TABLE 1 synthesized sense strand synthesized antisense strand sequencesequence duplex ID SS ID (5′-3′) AS ID (5′-3′) XD-00388 X01324GCGCUCGCCGCACGAGGCCdTdT X01325 GGCCUCGUGCGGCGAGCGCdTdT XD-00389 X01326CUUCGUGGGCCCGCGAUGCdTdT X01327 GCAUCGCGGGCCCACGAAGdTdT XD-00390 X01328AAGAACGCCGGGACAUGCCdTdT X01329 GGCAUGUCCCGGCGUUCUUdTdT XD-00391 X01330CAUGCCACGUGGUGGACCGdTdT X01331 CGGUCCACCACGUGGCAUGdTdT XD-00392 X01332CGGAGUACAAGUGCCGCUGdTdT X01333 CAGCGGCACUUGUACUCCGdTdT XD-00393 X01334UGCCGGCAGGAUGUCAACGdTdT X01335 CGUUGACAUCCUGCCGGCAdTdT XD-00394 X01336GAGGGUGUGCACUGCGAGGdTdT X01337 CCUCGCAGUGCACACCCUCdTdT XD-00395 X01338GGACCCAACACUUACACCUdTdT X01339 AGGUGUAAGUGUUGGGUCCdTdT XD-00396 X01340CUGCAAGGACGGCGUCGCCdTdT X01341 GGCGACGCCGUCCUUGCAGdTdT XD-00397 X01342GCACGUGUAUUGACGACGUdTdT X01343 ACGUCGUCAAUACACGUGCdTdT XD-00398 X01344CACGUGUAUUGACGACGUUdTdT X01345 AACGUCGUCAAUACACGUGdTdT XD-00399 X01346ACGUGUAUUGACGACGUUGdTdT X01347 CAACGUCGUCAAUACACGUdTdT XD-00400 X01348GGACGAGUGCUCACCCAGCdTdT X01349 GCUGGGUGAGCACUCGUCCdTdT XD-00401 X01350CCAUCAAGCGUGCCGCGGAdTdT X01351 UCGGCGGCACGCUUGAUGGdTdT XD-00402 X01352CCGGUUCGAGGAGCCCGUGdTdT X01353 CACGGGCUCCUCGAACCGGdTdT XD-00403 X01354CCGGGACAUCACGGAUCAUdTdT X01355 AUGAUCCGUGAUGUCCCGGdTdT XD-00404 X01356GACAUCGCACAGGAGCGCAdTdT X01357 UGCGCUCCUGUGCGAUGUCdTdT XD-00405 X01358CAGAGCGGCAUGGUGCCGAdTdT X01359 UCGGCACCAUGCCGCUCUGdTdT XD-00406 X01360CAUGGUGCCGAACCAAUACdTdT X01361 GUAUUGGUUCGGCACCAUGdTdT XD-00407 X01362UGGUGCCGAACCAAUACAAdTdT X01363 UUGUAUUGGUUCGGCACCAdTdT XD-00408 X01364CUCGCCUGUGGACAACACCdTdT X01365 GGUGUUGUCCACAGGCGAGdTdT XD-00409 X01366GACCAGUGGUCCAGCUCGDdTdT X01367 ACGAGCUGGACCACUGGUCdTdT XD-00410 X01368CAUUCCAACGUCUCCGACUdTdT X01369 AGUCGGAGACGUUGGAAUGdTdT XD-00411 X01370AUUCCAACGUCUCCGACUGdTdT X01371 CAGUCGGAGACGUUGGAAUdTdT XD-00412 X01372UUCCAACGUCUCCGACUGGdTdT X01373 CCAGUCGGAGACGUUGGAAdTdT XD-00413 X01374CAACGUCUCCGACUGGUCCdTdT X01375 GGACCAGUCGGAGACGUUGdTdT XD-00414 X01376ACGUCUCCGACUGGUCCGAdTdT X01377 UCGGACCAGUCGGAGACGUdTdT

The above Table 1 is represented again as Table 1a including thesequence identifiers.

TABLE 1a synthesized sense strand synthesized antisense strand duplex IDSS ID sequence (5′-3′) AS ID sequence (5′-3′) XD-00388 X01324GCGCUCGCCGCACGAGGCCdTdT X01325 GGCCUCGUGCGGCGAGCGCdTdT (SEQ ID NO: 73)(SEQ ID NO: 74) XD-00389 X01326 CUUCGUGGGCCCGCGAUGCdTdT X01327GCAUCGCGGGCCCACGAAGdTdT (SEQ ID NO: 75) (SEQ ID NO: 76) XD-00390 X01328AAGAACGCCGGGACAUGCCdTdT X01329 GGCAUGUCCCGGCGUUCUUdTdT (SEQ ID NO: 77)(SEQ ID NO: 78) XD-00391 X01330 CAUGCCACGUGGUGGACCGdTdT X01331CGGUCCACCACGUGGCAUGdTdT (SEQ ID NO: 79) (SEQ ID NO: 80) XD-00392 X01332CGGAGUACAAGUGCCGCUGdTdT X01333 CAGCGGCACUUGUACUCCGdTdT (SEQ ID NO: 81)(SEQ ID NO: 82) XD-00393 X01334 UGCCGGCAGGAUGUCAACGdTdT X01335CGUUGACAUCCUGCCGGCAdTdT (SEQ ID NO: 83) (SEQ ID NO: 84) XD-00394 X01336GAGGGUGUGCACUGCGAGGdTdT X01337 CCUCGCAGUGCACACCCUCdTdT (SEQ ID NO: 85)(SEQ ID NO: 86) XD-00395 X01338 GGACCCAACACUUACACCUdTdT X01339AGGUGUAAGUGUUGGGUCCdTdT (SEQ ID NO: 87) (SEQ ID NO: 88) XD-00396 X01340CUGCAAGGACGGCGUCGCCdTdT X01341 GGCGACGCCGUCCUUGCAGdTdT (SEQ ID NO: 89)(SEQ ID NO: 90) XD-00397 X01342 GCACGUGUAUUGACGACGUdTdT X01343ACGUCGUCAAUACACGUGCdTdT (SEQ ID NO: 91) (SEQ ID NO: 92) XD-00398 X01344CACGUGUAUUGACGACGUUdTdT X01345 AACGUCGUCAAUACACGUGdTdT (SEQ ID NO: 93)(SEQ ID NO: 94) XD-00399 X01346 ACGUGUAUUGACGACGUUGdTdT X01347CAACGUCGUCAAUACACGUdTdT (SEQ ID NO: 95) (SEQ ID NO: 96) XD-00400 X01348GGACGAGUGCUCACCCAGCdTdT X01349 GCUGGGUGAGCACUCGUCCdTdT (SEQ ID NO: 97)(SEQ ID NO: 98) XD-00401 X01350 CCAUCAAGCGUGCCGCCGAdTdT X01351UCGGCGGCACGCUUGAUGGdTdT (SEQ ID NO: 99) (SEQ ID NO: 100) XD-00402 X01352CCGGUUCGAGGAGCCCGUGdTdT X01353 CACGGGCUCCUCGAACCGGdTdT (SEQ ID NO: 101)(SEQ ID NO: 102) XD-00403 X01354 CCGGGACAUCACGGAUCAUdTdT X01355AUGAUCCGUGAUGUCCCGGdTdT (SEQ ID NO: 103) (SEQ ID NO: 104) XD-00404X01356 GACAUCGCACAGGAGCGCAdTdT X01357 UGCGCUCCUGUGCGAUGUCdTdT (SEQ IDNO: 105) (SEQ ID NO: 106) XD-00405 X01358 CAGAGCGGCAUGGUGCCGAdTdT X01359UCGGCACCAUGCCGCUCUGdTdT (SEQ ID NO: 107) (SEQ ID NO: 108) XD-00406X01360 CAUGGUGCCGAACCAAUACdTdT X01361 GUAUUGGUUCGGCACCAUGdTdT (SEQ IDNO: 109) (SEQ ID NO: 110) XD-00407 X01362 UGGUGCCGAACCAAUACAAdTdT X01363UUGUAUUGGUUCGGCACCAdTdT (SEQ ID NO: 111) (SEQ ID NO: 112) XD-00408X01364 CUCGCCUGUGGACAACACCdTdT X01365 GGUGUUGUCCACAGGCGAGdTdT (SEQ IDNO: 113) (SEQ ID NO: 114) XD-00409 X01366 GACCAGUGGUCCAGCUCGUdTdT X01367ACGAGCUGGACCACUGGUCdTdT (SEQ ID NO: 115) (SEQ ID NO: 116) XD-00410X01368 CAUUCCAACGUCUCCGACUdTdT X01369 AGUCGGAGACGUUGGAAUGdTdT (SEQ IDNO: 117) (SEQ ID NO: 118) XD-00411 X01370 AUUCCAACGUCUCCGACUGdTdT X01371CAGUCGGAGACGUUGGAAUdTdT (SEQ ID NO: 119) (SEQ ID NO: 120) XD-00412X01372 UUCCAACGUCUCCGACUGGdTdT X01373 CCAGUCGGAGACGUUGGAAdTdT (SEQ IDNO: 121) (SEQ ID NO: 122) XD-00413 X01374 CAACGUCUCCGACUGGUCCdTdT X01375GGACCAGUCGGAGACGUUGdTdT (SEQ ID NO: 123) (SEQ ID NO: 124) XD-00414X01376 ACGUCUCCGACUGGUCCGAdTdT X01377 UCGGACCAGUCGGAGACGUdTdT (SEQ IDNO: 125) (SEQ ID NO: 126)

Out of this group the following some siRNAs were modified. The modifiedsiRNAs are indicated in Table 2.

TABLE 2 a) XD-00395 sense strand: 5′ GGACCCAACACUUACACCUdTdT 3′ (SEQ IDNO: 87) antisense strand: 5′ AGGUGUAAGUGUUGGGUCCdTdT 3′ (SEQ ID NO: 88)b) XD-00404 sense strand: 5′ GACAUCGCACAGGAGCGCAdTdT 3′ (SEQ ID NO: 105)antisense strand: 5′ UGCGCUCCUGUGCGAUGUCdTdT 3′ (SEQ ID NO: 106) c)XD-00406 sense strand: 5′ CAUGGUGCCGAACCAAUACdTdT 3′ (SEQ ID NO: 109)antisense strand: 5′ GUAUUGGUUCGGCACCAUGdTdT 3′ (SEQ ID NO: 110) d)XD-00407 sense strand: 5′ UGGUGCCGAACCAAUACAAdTdT 3′ (SEQ ID NO: 111)antisense strand: 5′ UUGUAUUGGUUCGGCACCAdTdT 3′ (SEQ ID NO: 112) e)XD-00409 sense strand: 5′ GACCAGUGGUCCAGCUCGUdTdT 3′ (SEQ ID NO: 115)antisense strand: 5′ ACGAGCUGGACCACUGGUCdTdT 3′ (SEQ ID NO: 116) f)XD-00410 sense strand: 5′ CAUUCCAACGUCUCCGACUdTdT 3′ (SEQ ID NO: 117)antisense strand: 5′ AGUCGGAGACGUUGGAAUGdTdT 3′ (SEQ ID NO: 118)Fully Stabilized siRNA

siRNAs XD-00404 and XD-00409 of Table 2 were subject to fullstabilization. The thus fully stabilized siRNAs are as follow.

a) XD-00409 sense strand: (SEQ ID NO: 70) 5′ GAcCaGuGgUcCaGcUcGudTsdT 3′antisense strand: (SEQ ID NO: 69) 5′ acGaGcUgGaCcAcUgGuCdTsdT 3′ b)XD-00404 sense strand: (SEQ ID NO: 128) 5′ GAcAuCgCaCaGgAgCgCadTsdT 3′antisense strand: (SEQ ID NO: 127) 5′ ugCgCuCcUgUgCgAuGuCdTsdT 3′,wherein a minor nucleotide indicates that the nucleotide is 2′-Fmodified and an underlined nucleotide indicates that the nucleotide is2′-O-methyl modified andwherein dTsdT indicates that at the 3′ end a dinucleotide is attachedconsisting of two dTs, wherein said two dTs are covalently linkedthrough a phosphorothioate bondIntermediately Stabilized siRNA

The intermediately stabilized siRNAs differ from the fully stabilizedsiRNAs such that lack the dTsdT overhang and did not exhibit any 2′-Fmodification.

Cultivation of C4-2 Cells

C4-2 cells were cultivated according to standard procedures describedfor this cell line using RPMI 1640 medium.

Transfection of C4-2 Cells

C4-2 cells were transfected with various concentrations of siRNA usingLipofectamine 2000. Concentrations of siRNA were 50 nM, l OnM, 1 nM or0.1 nM in the transfection experiment. Incubation time was 24 h.Otherwise a standard protocol was used. Transfection efficiency wasdetermined by measuring housekeeper siRNA; transfection efficacy was atleast 93% in all cases.

Determining the IC 50 of siRNAs

IC50 values were determined using standard procedures. Morespecifically, siRNA concentration was determined at which expression ofNotch 1 mRNA was decreased to 50% using C4-2 cells transfected asdescribed using the various siRNAs indicated.

Nanocarrier

For preparation of the nanocarrier, which is a perfluorocarbonnanocarrier, perfluoroocytlbromide (Perflubron) was emulsified with amixture of phospholipids. One gram of the mixture containsphosphatidylcholine (980 mg), sphingomyelin (10 mg), cholesterol (5 mg),Iysophoshatidylcholine (5 mg), in distilled water and 75 mM sodiumdihydrogen phosphate (NaH₂PO₄) buffer. To gain 1000 μl of theperfluorcarbon nanocarrier, 475 μl perfluorooctylbromide, 36 mgphospholipids, 200 μl 75 mM NaH₂PO₄ at pH 7.4 and 325 μl distilled waterwas used.

First, phospholipids, sodium dihydrogen phosphate buffer and distilledwater were mixed and subsequently the perfluorcarbon (PFC) solution wasadjoined. Within 40 seconds, the composite had to be mixed by a shakerfor 60 s and without any interruption homogenized twice by an ultrasonicdevice at a frequency of 1100 kHz for 120 s with intervals of 30 s. Thesonication unit is kept at a temperature of 4° C. For the final emulsionof the otherwise insoluble PFC, the mixture is given into a highpressure homogenizer. Within six passages of homogenization at 2500 barthe milky composite turns into a transparent, bluish emulsion. Thischange to transparency is a macroscopic marker for the turn of theperfluorcarbon particles size below the visible wavelengths. The lowestvisible wavelength (blue/violet) of λ=400 nm defines the particles sizeas λ/2 when the mixture becomes transparent. Four additional cycles ofhomogenization are added at this point. The particles size was measuredin electron microscopy as 50 nm (mean) with all particles below 100 nm.To gain the functional nanocarrier, 4 mg holotransferrin is solved in 60μl sterile 0.9% NaCl. Directly afterwards, the transferrin ishomogenized for 2 s by the cooled ultrasonic device. The solvedtransferrin is added to 1000 μl perfluorocarbon emulsion to obtain anend concentration of 4 mg/ml. Again, the compound is directly put on ashaker for 30 s.

This nanocarrier is also referred to as unloaded carrier or NCf4.

Notch siRNA-Loaded Nanocarriers

Notch siRNA-loaded nanocarriers were prepared based on the Nanocarrierdescribed above. For loading purposes the desired siRNA species wasadded to the nanocarriersand the thus obtained mixture subject tohomogenization by ultrasound using 500 W for 15 s.

Animal Study—Xenograft Model

Sixty female athymic nude Foxn1^(nu) mice bearing tumours fromsubcutaneously inoculated PANC-1 human pancreatic tumours were selectedfrom a pool of 112. This animal model is an established animal model forpancreatic cancer and pancreatic tumor, respectively, using PANC-1 cellline which has been first described by Lieber M et al. (M. Lieber, etal., “Establishment of a continuous tumor-cell line (panc-1) from ahuman carcinoma of the exocrine pancreas,” Int J. Cancer 15(5), 741(1975)), and biochemically as well as morphologically characterized byMadden M E and Sarras M P (M. E. Madden and M. P. Sarras, Jr.,“Morphological and biochemical characterization of a human pancreaticductal cell line (PANC-1),” Pancreas 3(5), 512 (1988)). Such cell lineand the established animal model using such cell line has been used inthe evaluation of anti-cancer agents Schultz R M et al. (R. M. Schultz,et al., “Evaluation of new anticancer agents against the MIA PaCa-2 andPANC-1 human pancreatic carcinoma xenografts,” Oncol. Res. 5(6-7), 223(1993)).

Thirty-three days post-inoculation, the mice were randomised by tumoursize into six groups of ten (Day 0).

Mice in each group were treated twice weekly with Vehicle Control(unloaded nanocarrier, NCf4) or Gemcitabine at 60 mg/kg viaintraperitoneal injection, or a combination of Notch/Gemcitabine, eachin the manner described, whereby Notch was Notch siRNA-loadednanocarriers (prepared as described in Example 1 using fully stabilizedXD-00409) treated via intravenous injection.

Treatments commenced on Day 0 and seven doses were administered.Clinical observations were made daily. Body weight and tumour sizemeasurements were made three times weekly for the duration of the study.

Upon termination of the study (Day 24), tumours were harvested from allmice in all treatment groups, weighed and cut in half. One portion waspreserved in RNAlater solution for isolation of RNA and qRT-PCRanalysis. The remaining portion was preserved in formalin for paraffinembedding and microscopic assessment of necrosis.

Animal Study—Orthotopic Model

Sixty-six female athymic nude Foxn1^(nu) mice were orthotopicallyinoculated with PANC-1 human pancreatic tumour cells. Take-rate wasassessed in three mice each on Days 20 and 27 post-inoculation.Thirty-two days post-inoculation, the remaining 60 mice were randomisedby body weight into six groups of ten (Day 0).

Mice in each group were treated twice weekly with Vehicle Control(unloaded nanocarrier, NCf4) Gemcitabine at 60 mg/kg via intraperitonealinjection, or a combination of Notch/Gemcitabine, each in the mannerdescribed, whereby Notch was Notch siRNA-loaded nanocarriers (preparedas described in Example 1 using fully stabilized XD-00409) treated viaintravenous injection.

Treatments commenced on Day 0 and five doses were administered.

Clinical observations were made daily. Body weight measurements weremade three times weekly for the duration of the study.

Upon termination of the study (Day 18), the intact tumor+ pancreas washarvested from all mice in all treatment groups and weighed. Tumors wereremoved from the pancreas and cut in half. One portion was preserved inRNAlater solution for optional isolation of RNA and qRT-PCR analysis(not performed). The remaining portion was preserved in formalin forparaffin embedding and microscopic assessment of necrosis. The lungs andliver were harvested from all mice at termination. Both were weighed andassessed for surface metastases. The lungs and livers were preserved informalin for paraffin embedding. Livers were assessed for the presenceof micro-metastases.

EXAMPLE 2 Efficacy of Non-Modified siRNA Targeting Human Notch 1

C4-2 cells were cultivated and transfected with the various siRNAsindicated in Table 1 as described in Example 1, whereby siRNAconcentration was 50 nM. The results are shown in FIG. 1. FIG. 1 is adiagram indicating the relative expression of Notch 1 upon transfectionof C4-2 cells using various siRNAs. Expression is normalized to theexpression of Notch 1 in C4-2 cells using a control. Control was a siRNAwhich is did not target any known mRNA coding for a protein.

As may be taken from FIG. 1 the best siRNAs show a knockdown of Notch 1mRNA of almost 80%. It is also evident from FIG. 1 that a significantdifference in efficacy of the various siRNAs exists.

The 6 siRNAs showing best knock-down are the ones of Table 2. ThesesiRNAs were characterized further in terms of their IC50. The result isshown in FIG. 2

FIG. 2 is a diagram for each siRNA of Table 2 indicating the remainingrelative Notch 1 mRNA expression as a function of the concentration ofeach siRNA. From said diagrams the IC50 for each of said siRNAs wascalculated. The IC 50 values for said siRNA molecules are summarized inTable 3.

TABLE 3 siRNA IC₅₀ XD-00409 0.0043 XD-00404 0.042 XD-00410 0.061XD-00407 0.073 XD-00395 0.111 XD-00406 0.545

As may be taken from both FIG. 2 and Table 3 the best siRNA molecule interms of IC50 is XD-00409 having an IC50 of 4.3 pM.

EXAMPLE 3 Efficacy of Modified siRNA Targeting Human Notch 1

siRNA molecules XD00404 and XD-00409 of Tables 1 and 2 were subject tointermediate stabilization and full stabilization as also described inExample 1. The accordingly modified siRNA molecules are as follows

a) XD-00404 with intermediate stabilization (also referred to asXD-00751):b) XD-00404 with full stabilization (also referred to as XD-00752):c) XD-00409 with intermediate stabilization (also referred to asXD-00753):d) XD-00409 with full stabilization (also referred to as XD-00754):

These siRNA molecules were tested as to their efficacy in C4-2 cellsupon transfection of said C4-2 cells as described in Example 1, wherebythe concentration of the siRNA was 10 nM, 1 nM or 0.1 nM in thetransfection experiment.

The results of such experiments are shown in FIG. 3A for siRNA XD-00404in its unmodified, intermediate or fully modified form and for siRNAXD-00409 in its unmodified, intermediate or fully modified form and inFIG. 3B as relative mRNA expression using a control. Control was a siRNAwhich is did not target any known mRNA coding for a protein.

As may be taken from FIG. 3, although modification of the siRNA moleculeis beneficial in terms of stability, it is evident that modification ofboth siRNAs (XD-00404 and XD-00409) is reducing its efficacy of theknock-down of the expression of the Notch 1 mRNA. It has, however,surprisingly found that the impact of modification on XD-00409 is lesspronounced and factually not existing compared to the impact ofmodification on XD-00404. Insofar, siRNA DX-00409 shows surprising andunexpected effects.

EXAMPLE 4 Effect of siRNA Targeting Human Notch 1 in a XenograftPancreas Tumor Model

The animal study using PANC-1 human pancreatic tumours as described inExample 1 was carried out. The siRNA species used was fully stabilizedXD-00409.

The results of the study can be summarized as follows:

-   -   Body weight loss was not evident. A small mass was present under        the front leg in a total of 10 mice in the study.    -   Mild tumour inhibition was evident following treatment with        Gemcitabine monotherapy, and combination of Notch/Gemcitabine,        indicated by measurements of percentage tumour growth and tumour        weight, but only Notch/Gemcitabine combination therapy was        significantly different to the Vehicle in regards to tumour        weight. A synergistic response in tumour inhibition, measured by        percentage tumour growth, was exhibited by combination therapy        Notch/Gemcitabine.    -   Mice treated with Gemcitabine monotherapy and Notch/Gemcitabine        combination therapy had the lowest occurrence of tumour        necrosis.

The result of said animal study is also shown in FIG. 4. FIG. 4 is adiagram showing the relative increase in tumour volume (indicated as%±SEM) over time using control (“Vehicle (NCF4)”), gemcitabine(“Gemcitabine”) and a combination of gemcitabine and the NotchsiRNA-loaded nanocarrier (“Notch/Gemcitabine”). Each of the agents orcombination agent was administered 2× weekly.

EXAMPLE 5 Effect of siRNA Targeting Human Notch 1 in an OrthotopicPancreatic Tumor Model

The animal study using PANC-1 human pancreatic tumours as described inExample 1 was carried out. The siRNA species used was fully stabilizedXD-00409.

The results of the study can be summarized as follows:

The number of animals without metastases based on PANC-1 cells wassignificantly reduced as shown in FIG. 5 in case of the animals whichreceived a combination of gemcitabine and the Notch siRNA-loadednanocarrier compared to the animals receiving either control or onlygemcitabine. More specifically, upon treatment of ten animals withvehicle alone, three animals had several metastases, three animals hadsingle metastasis and four animals were without metastases; upontreatment of ten animals with Gemcitabine alone, two animals had severalmetastases, four animals had single metastasis and four animals werewithout metastases; and upon treatment of ten animals with bothgemcitabine and siRNA-loaded nanocarriers using fully modified XD-00409,one animal had several metastases, one animal had single metastasis andeight animals were without metastases.

Furthermore, treatment with Gemcitabine in combination with Notch 1specific siRNA (fully stabilized XD-00409), was associated withsignificant mean body weight loss and with loss of body condition in twomice.

The content and disclosure of the various references recited herein isincorporated herein by reference in their entirety.

The features of the present invention disclosed in the specification,the claims and/or the drawings may both separately and in anycombination thereof be material for realizing the invention in variousforms thereof.

1. A nucleic acid molecule comprising a double-stranded structure,wherein the double-stranded structure is formed by a first strand and asecond strand, wherein the first strand consist of the followingnucleotide sequence 5′ acGaGcUgGaCcAcUgGuCdTsdT 3′,

and the second strand consists of the following nucleotide sequence5′ GAcCaGuGgUcCaGcUcGudTsdT 3′,

wherein a minor nucleotide indicates that the nucleotide is 2′-Fmodified and an underlined nucleotide indicates that the nucleotide is2′-0-methyl modified and wherein dTsdT indicates that at the 3′ end adinucleotide is attached consisting of two dT nucleotides, wherein saidtwo dTs are covalently linked through a phosphorothioate bond. 2-14.(canceled)
 15. The nucleic acid molecule of claim 1, wherein the nucleicacid molecule is capable of causing post-transcriptional silencing of agene.
 16. The nucleic acid molecule of claim 15, wherein the gene ishuman Notch
 1. 17. The nucleic acid molecule of claim 1, whereinpost-transcriptional silencing is RNA interference.
 18. The nucleic acidmolecule of claim 15, wherein post-transcriptional silencing is RNAinterference.
 19. The nucleic acid molecule of claim 16, whereinpost-transcriptional silencing is RNA interference.
 20. The nucleic acidmolecule of claim 1, for use in a method for the treatment and/orprevention of a disease, wherein the method comprises administering to asubject the nucleic acid molecule, preferably the disease is selectedfrom the group comprising esophageal cancer, oral squamous cellcarcinoma, head and neck cancer, tongue cancer, leukemia, renal cellcarcinoma, gastric cancer, colon adenocarcinoma, endometrialcancer/uterine corpus, cervical cancer/uterine cervix, intrahepaticcholangiocarcinoma, hepatocellular carcinoma, osteosarcoma, urinarybladder carcinoma, malignant melanoma, thyroid cancer, lungadenocarcinoma, prostate cancer, breast cancer, ovarian cancer,pancreatic cancer and glioma.
 21. The nucleic acid molecule of claim 15,for use in a method for the treatment and/or prevention of a disease,wherein the method comprises administering to a subject the nucleic acidmolecule, preferably the disease is selected from the group comprisingesophageal cancer, oral squamous cell carcinoma, head and neck cancer,tongue cancer, leukemia, renal cell carcinoma, gastric cancer, colonadenocarcinoma, endometrial cancer/uterine corpus, cervicalcancer/uterine cervix, intrahepatic cholangiocarcinoma, hepatocellularcarcinoma, osteosarcoma, urinary bladder carcinoma, malignant melanoma,thyroid cancer, lung adenocarcinoma, prostate cancer, breast cancer,ovarian cancer, pancreatic cancer and glioma.
 22. The nucleic acidmolecule of claim 16, for use in a method for the treatment and/orprevention of a disease, wherein the method comprises administering to asubject the nucleic acid molecule, preferably the disease is selectedfrom the group comprising esophageal cancer, oral squamous cellcarcinoma, head and neck cancer, tongue cancer, leukemia, renal cellcarcinoma, gastric cancer, colon adenocarcinoma, endometrialcancer/uterine corpus, cervical cancer/uterine cervix, intrahepaticcholangiocarcinoma, hepatocellular carcinoma, osteosarcoma, urinarybladder carcinoma, malignant melanoma, thyroid cancer, lungadenocarcinoma, prostate cancer, breast cancer, ovarian cancer,pancreatic cancer and glioma.
 23. The nucleic acid molecule of claim 17,for use in a method for the treatment and/or prevention of a disease,wherein the method comprises administering to a subject the nucleic acidmolecule, preferably the disease is selected from the group comprisingesophageal cancer, oral squamous cell carcinoma, head and neck cancer,tongue cancer, leukemia, renal cell carcinoma, gastric cancer, colonadenocarcinoma, endometrial cancer/uterine corpus, cervicalcancer/uterine cervix, intrahepatic cholangiocarcinoma, hepatocellularcarcinoma, osteosarcoma, urinary bladder carcinoma, malignant melanoma,thyroid cancer, lung adenocarcinoma, prostate cancer, breast cancer,ovarian cancer, pancreatic cancer and glioma.
 24. The nucleic acidmolecule of claim 18, for use in a method for the treatment and/orprevention of a disease, wherein the method comprises administering to asubject the nucleic acid molecule, preferably the disease is selectedfrom the group comprising esophageal cancer, oral squamous cellcarcinoma, head and neck cancer, tongue cancer, leukemia, renal cellcarcinoma, gastric cancer, colon adenocarcinoma, endometrialcancer/uterine corpus, cervical cancer/uterine cervix, intrahepaticcholangiocarcinoma, hepatocellular carcinoma, osteosarcoma, urinarybladder carcinoma, malignant melanoma, thyroid cancer, lungadenocarcinoma, prostate cancer, breast cancer, ovarian cancer,pancreatic cancer and glioma.
 25. The nucleic acid molecule of claim 20,wherein the method comprises administering a further pharmaceuticallyactive agent to the subject.
 26. The nucleic acid molecule of claim 21,wherein the method comprises administering a further pharmaceuticallyactive agent to the subject.
 27. The nucleic acid molecule of claim 22,wherein the method comprises administering a further pharmaceuticallyactive agent to the subject.
 28. The nucleic acid molecule of claim 23,wherein the method comprises administering a further pharmaceuticallyactive agent to the subject.
 29. The nucleic acid molecule of claim 24,wherein the method comprises administering a further pharmaceuticallyactive agent to the subject.
 30. A method for restoring drug sensitivityof cancer cells by administering the nucleic acid molecule of claim 1,wherein the cancer cells exhibit Notch 1 induced chemoresistance.
 31. Amethod for restoring drug sensitivity of cancer cells by administeringthe nucleic acid molecule of claim 16, wherein the cancer cells exhibitNotch 1 induced chemoresistance.
 32. The nucleic acid molecule of claim16, for use in a method for restoring drug sensitivity of cancer cells,wherein the cancer cells exhibit Notch 1 induced chemoresistance.
 33. Ananoemulsion comprising a discontinuous phase and a continuous aqueousphase and a nucleic acid molecule of claim
 1. 34. A nanoemulsioncomprising a discontinuous phase and a continuous aqueous phase and anucleic acid molecule of claim
 15. 35. A nanoemulsion comprising adiscontinuous phase and a continuous aqueous phase and a nucleic acidmolecule of claim
 16. 36. The nanoemulsion of claim 33, wherein thediscontinuous phase comprises a perfluorocarbon phase.
 37. Thenanoemulsion of claim 34, wherein the discontinuous phase comprises aperfluorocarbon phase.
 38. The nanoemulsion of claim 35, wherein thediscontinuous phase comprises a perfluorocarbon phase.
 39. Thenanoemulsion of claim 33, wherein the nanoemulsion comprises anendocytosis enhancing surface, preferably the endocytosis enhancingsurface comprises an endocytosis enhancing component, wherein theendocytosis enhancing component is selected from the group comprising atleast one compound inducing cellular uptake of the nanoemulsion orparticles of the nanoemulsion via endocytosis.
 40. The nanoemulsion ofclaim 34, wherein the nanoemulsion comprises an endocytosis enhancingsurface, preferably the endocytosis enhancing surface comprises anendocytosis enhancing component, wherein the endocytosis enhancingcomponent is selected from the group comprising at least one compoundinducing cellular uptake of the nanoemulsion or particles of thenanoemulsion via endocytosis.
 41. The nanoemulsion of claim 35, whereinthe nanoemulsion comprises an endocytosis enhancing surface, preferablythe endocytosis enhancing surface comprises an endocytosis enhancingcomponent, wherein the endocytosis enhancing component is selected fromthe group comprising at least one compound inducing cellular uptake ofthe nanoemulsion or particles of the nanoemulsion via endocytosis. 42.The nanoemulsion of claim 36, wherein the nanoemulsion comprises anendocytosis enhancing surface, preferably the endocytosis enhancingsurface comprises an endocytosis enhancing component, wherein theendocytosis enhancing component is selected from the group comprising atleast one compound inducing cellular uptake of the nanoemulsion orparticles of the nanoemulsion via endocytosis.
 43. The nanoemulsion ofclaim 37, wherein the nanoemulsion comprises an endocytosis enhancingsurface, preferably the endocytosis enhancing surface comprises anendocytosis enhancing component, wherein the endocytosis enhancingcomponent is selected from the group comprising at least one compoundinducing cellular uptake of the nanoemulsion or particles of thenanoemulsion via endocytosis.
 44. The nanoemulsion of claim 38, whereinthe nanoemulsion comprises an endocytosis enhancing surface, preferablythe endocytosis enhancing surface comprises an endocytosis enhancingcomponent, wherein the endocytosis enhancing component is selected fromthe group comprising at least one compound inducing cellular uptake ofthe nanoemulsion or particles of the nanoemulsion via endocytosis. 45.The nanoemulsion of claim 39, wherein the nanoemulsion comprises anendocytosis enhancing surface, preferably the endocytosis enhancingsurface comprises an endocytosis enhancing component, wherein theendocytosis enhancing component is selected from the group comprising atleast one compound inducing cellular uptake of the nanoemulsion orparticles of the nanoemulsion via endocytosis.
 46. The nanoemulsion ofclaim 40, wherein the nanoemulsion comprises an endocytosis enhancingsurface, preferably the endocytosis enhancing surface comprises anendocytosis enhancing component, wherein the endocytosis enhancingcomponent is selected from the group comprising at least one compoundinducing cellular uptake of the nanoemulsion or particles of thenanoemulsion via endocytosis.
 47. The nanoemulsion of claim 41, whereinthe nanoemulsion comprises an endocytosis enhancing surface, preferablythe endocytosis enhancing surface comprises an endocytosis enhancingcomponent, wherein the endocytosis enhancing component is selected fromthe group comprising at least one compound inducing cellular uptake ofthe nanoemulsion or particles of the nanoemulsion via endocytosis. 48.The nanoemulsion of claim 42, wherein the nanoemulsion comprises anendocytosis enhancing surface, preferably the endocytosis enhancingsurface comprises an endocytosis enhancing component, wherein theendocytosis enhancing component is selected from the group comprising atleast one compound inducing cellular uptake of the nanoemulsion orparticles of the nanoemulsion via endocytosis.
 49. The nanoemulsion ofclaim 43, wherein the nanoemulsion comprises an endocytosis enhancingsurface, preferably the endocytosis enhancing surface comprises anendocytosis enhancing component, wherein the endocytosis enhancingcomponent is selected from the group comprising at least one compoundinducing cellular uptake of the nanoemulsion or particles of thenanoemulsion via endocytosis.
 50. The nanoemulsion of claim 44, whereinthe nanoemulsion comprises an endocytosis enhancing surface, preferablythe endocytosis enhancing surface comprises an endocytosis enhancingcomponent, wherein the endocytosis enhancing component is selected fromthe group comprising at least one compound inducing cellular uptake ofthe nanoemulsion or particles of the nanoemulsion via endocytosis. 51.The nanoemulsion of claim 45, wherein the nanoemulsion comprises anendocytosis enhancing surface, preferably the endocytosis enhancingsurface comprises an endocytosis enhancing component, wherein theendocytosis enhancing component is selected from the group comprising atleast one compound inducing cellular uptake of the nanoemulsion orparticles of the nanoemulsion via endocytosis.
 52. The nanoemulsion ofclaim 46, wherein the nanoemulsion comprises an endocytosis enhancingsurface, preferably the endocytosis enhancing surface comprises anendocytosis enhancing component, wherein the endocytosis enhancingcomponent is selected from the group comprising at least one compoundinducing cellular uptake of the nanoemulsion or particles of thenanoemulsion via endocytosis.
 53. A method of using the nanoemulsion ofclaim 33, for use in a method for the treatment and/or prevention of adisease, wherein the method comprises administering to a subject thenucleic acid molecule, preferably the disease is selected from the groupcomprising esophageal cancer, oral squamous cell carcinoma, head andneck cancer, tongue cancer, leukemia, renal cell carcinoma, gastriccancer, colon adenocarcinoma, endometrial cancer/uterine corpus,cervical cancer/uterine cervix, intrahepatic cholangiocarcinoma,hepatocellular carcinoma, osteosarcoma, urinary bladder carcinoma,malignant melanoma, thyroid cancer, lung adenocarcinoma, prostatecancer, breast cancer, ovarian cancer, pancreatic cancer and glioma. 54.A method of using the nanoemulsion of claim 33, for use in a method forthe treatment and/or prevention of a disease, wherein the methodcomprises administering to a subject the nucleic acid molecule,preferably the disease is selected from the group comprising esophagealcancer, oral squamous cell carcinoma, head and neck cancer, tonguecancer, leukemia, renal cell carcinoma, gastric cancer, colonadenocarcinoma, endometrial cancer/uterine corpus, cervicalcancer/uterine cervix, intrahepatic cholangiocarcinoma, hepatocellularcarcinoma, osteosarcoma, urinary bladder carcinoma, malignant melanoma,thyroid cancer, lung adenocarcinoma, prostate cancer, breast cancer,ovarian cancer, pancreatic cancer and glioma.
 55. A method of using thenanoemulsion of claim 33, for use in a method for the treatment and/orprevention of a disease, wherein the method comprises administering to asubject the nucleic acid molecule, preferably the disease is selectedfrom the group comprising esophageal cancer, oral squamous cellcarcinoma, head and neck cancer, tongue cancer, leukemia, renal cellcarcinoma, gastric cancer, colon adenocarcinoma, endometrialcancer/uterine corpus, cervical cancer/uterine cervix, intrahepaticcholangiocarcinoma, hepatocellular carcinoma, osteosarcoma, urinarybladder carcinoma, malignant melanoma, thyroid cancer, lungadenocarcinoma, prostate cancer, breast cancer, ovarian cancer,pancreatic cancer and glioma.
 48. A method of using the nanoemulsion ofclaim 33, for use in a method for the treatment and/or prevention of adisease, wherein the method comprises administering to a subject thenucleic acid molecule, preferably the disease is selected from the groupcomprising esophageal cancer, oral squamous cell carcinoma, head andneck cancer, tongue cancer, leukemia, renal cell carcinoma, gastriccancer, colon adenocarcinoma, endometrial cancer/uterine corpus,cervical cancer/uterine cervix, intrahepatic cholangiocarcinoma,hepatocellular carcinoma, osteosarcoma, urinary bladder carcinoma,malignant melanoma, thyroid cancer, lung adenocarcinoma, prostatecancer, breast cancer, ovarian cancer, pancreatic cancer and glioma. 49.A method of using the nanoemulsion of claim 33, for use in a method forthe treatment and/or prevention of a disease, wherein the methodcomprises administering to a subject the nucleic acid molecule,preferably the disease is selected from the group comprising esophagealcancer, oral squamous cell carcinoma, head and neck cancer, tonguecancer, leukemia, renal cell carcinoma, gastric cancer, colonadenocarcinoma, endometrial cancer/uterine corpus, cervicalcancer/uterine cervix, intrahepatic cholangiocarcinoma, hepatocellularcarcinoma, osteosarcoma, urinary bladder carcinoma, malignant melanoma,thyroid cancer, lung adenocarcinoma, prostate cancer, breast cancer,ovarian cancer, pancreatic cancer and glioma.
 50. A method of using thenanoemulsion of claim 33, for use in a method for the treatment and/orprevention of a disease, wherein the method comprises administering to asubject the nucleic acid molecule, preferably the disease is selectedfrom the group comprising esophageal cancer, oral squamous cellcarcinoma, head and neck cancer, tongue cancer, leukemia, renal cellcarcinoma, gastric cancer, colon adenocarcinoma, endometrialcancer/uterine corpus, cervical cancer/uterine cervix, intrahepaticcholangiocarcinoma, hepatocellular carcinoma, osteosarcoma, urinarybladder carcinoma, malignant melanoma, thyroid cancer, lungadenocarcinoma, prostate cancer, breast cancer, ovarian cancer,pancreatic cancer and glioma.
 51. A method of using the nanoemulsion ofclaim 33, for use in a method for the treatment and/or prevention of adisease, wherein the method comprises administering to a subject thenucleic acid molecule, preferably the disease is selected from the groupcomprising esophageal cancer, oral squamous cell carcinoma, head andneck cancer, tongue cancer, leukemia, renal cell carcinoma, gastriccancer, colon adenocarcinoma, endometrial cancer/uterine corpus,cervical cancer/uterine cervix, intrahepatic cholangiocarcinoma,hepatocellular carcinoma, osteosarcoma, urinary bladder carcinoma,malignant melanoma, thyroid cancer, lung adenocarcinoma, prostatecancer, breast cancer, ovarian cancer, pancreatic cancer and glioma. 52.A method of using the nanoemulsion of claim 33, for use in a method forthe treatment and/or prevention of a disease, wherein the methodcomprises administering to a subject the nucleic acid molecule,preferably the disease is selected from the group comprising esophagealcancer, oral squamous cell carcinoma, head and neck cancer, tonguecancer, leukemia, renal cell carcinoma, gastric cancer, colonadenocarcinoma, endometrial cancer/uterine corpus, cervicalcancer/uterine cervix, intrahepatic cholangiocarcinoma, hepatocellularcarcinoma, osteosarcoma, urinary bladder carcinoma, malignant melanoma,thyroid cancer, lung adenocarcinoma, prostate cancer, breast cancer,ovarian cancer, pancreatic cancer and glioma.
 53. A method of using thenanoemulsion of claim 33, for use in a method for the treatment and/orprevention of a disease, wherein the method comprises administering to asubject the nucleic acid molecule, preferably the disease is selectedfrom the group comprising esophageal cancer, oral squamous cellcarcinoma, head and neck cancer, tongue cancer, leukemia, renal cellcarcinoma, gastric cancer, colon adenocarcinoma, endometrialcancer/uterine corpus, cervical cancer/uterine cervix, intrahepaticcholangiocarcinoma, hepatocellular carcinoma, osteosarcoma, urinarybladder carcinoma, malignant melanoma, thyroid cancer, lungadenocarcinoma, prostate cancer, breast cancer, ovarian cancer,pancreatic cancer and glioma.
 54. A method of using the nanoemulsion ofclaim 34, for use in a method for the treatment and/or prevention of adisease, wherein the method comprises administering to a subject thenucleic acid molecule, preferably the disease is selected from the groupcomprising esophageal cancer, oral squamous cell carcinoma, head andneck cancer, tongue cancer, leukemia, renal cell carcinoma, gastriccancer, colon adenocarcinoma, endometrial cancer/uterine corpus,cervical cancer/uterine cervix, intrahepatic cholangiocarcinoma,hepatocellular carcinoma, osteosarcoma, urinary bladder carcinoma,malignant melanoma, thyroid cancer, lung adenocarcinoma, prostatecancer, breast cancer, ovarian cancer, pancreatic cancer and glioma. 55.A method of using the nanoemulsion of claim 35, for use in a method forthe treatment and/or prevention of a disease, wherein the methodcomprises administering to a subject the nucleic acid molecule,preferably the disease is selected from the group comprising esophagealcancer, oral squamous cell carcinoma, head and neck cancer, tonguecancer, leukemia, renal cell carcinoma, gastric cancer, colonadenocarcinoma, endometrial cancer/uterine corpus, cervicalcancer/uterine cervix, intrahepatic cholangiocarcinoma, hepatocellularcarcinoma, osteosarcoma, urinary bladder carcinoma, malignant melanoma,thyroid cancer, lung adenocarcinoma, prostate cancer, breast cancer,ovarian cancer, pancreatic cancer and glioma.
 56. A method of using thenanoemulsion of claim 36, for use in a method for the treatment and/orprevention of a disease, wherein the method comprises administering to asubject the nucleic acid molecule, preferably the disease is selectedfrom the group comprising esophageal cancer, oral squamous cellcarcinoma, head and neck cancer, tongue cancer, leukemia, renal cellcarcinoma, gastric cancer, colon adenocarcinoma, endometrialcancer/uterine corpus, cervical cancer/uterine cervix, intrahepaticcholangiocarcinoma, hepatocellular carcinoma, osteosarcoma, urinarybladder carcinoma, malignant melanoma, thyroid cancer, lungadenocarcinoma, prostate cancer, breast cancer, ovarian cancer,pancreatic cancer and glioma.
 57. A method of using the nanoemulsion ofclaim 33, for use in a method for the treatment and/or prevention of adisease, wherein the method comprises administering to a subject thenucleic acid molecule, preferably the disease is selected from the groupcomprising esophageal cancer, oral squamous cell carcinoma, head andneck cancer, tongue cancer, leukemia, renal cell carcinoma, gastriccancer, colon adenocarcinoma, endometrial cancer/uterine corpus,cervical cancer/uterine cervix, intrahepatic cholangiocarcinoma,hepatocellular carcinoma, osteosarcoma, urinary bladder carcinoma,malignant melanoma, thyroid cancer, lung adenocarcinoma, prostatecancer, breast cancer, ovarian cancer, pancreatic cancer and glioma. 58.A method of using the nanoemulsion of claim 38, for use in a method forthe treatment and/or prevention of a disease, wherein the methodcomprises administering to a subject the nucleic acid molecule,preferably the disease is selected from the group comprising esophagealcancer, oral squamous cell carcinoma, head and neck cancer, tonguecancer, leukemia, renal cell carcinoma, gastric cancer, colonadenocarcinoma, endometrial cancer/uterine corpus, cervicalcancer/uterine cervix, intrahepatic cholangiocarcinoma, hepatocellularcarcinoma, osteosarcoma, urinary bladder carcinoma, malignant melanoma,thyroid cancer, lung adenocarcinoma, prostate cancer, breast cancer,ovarian cancer, pancreatic cancer and glioma.
 59. A method of using thenanoemulsion of claim 39, for use in a method for the treatment and/orprevention of a disease, wherein the method comprises administering to asubject the nucleic acid molecule, preferably the disease is selectedfrom the group comprising esophageal cancer, oral squamous cellcarcinoma, head and neck cancer, tongue cancer, leukemia, renal cellcarcinoma, gastric cancer, colon adenocarcinoma, endometrialcancer/uterine corpus, cervical cancer/uterine cervix, intrahepaticcholangiocarcinoma, hepatocellular carcinoma, osteosarcoma, urinarybladder carcinoma, malignant melanoma, thyroid cancer, lungadenocarcinoma, prostate cancer, breast cancer, ovarian cancer,pancreatic cancer and glioma.
 60. A method of using the nanoemulsion ofclaim 40, for use in a method for the treatment and/or prevention of adisease, wherein the method comprises administering to a subject thenucleic acid molecule, preferably the disease is selected from the groupcomprising esophageal cancer, oral squamous cell carcinoma, head andneck cancer, tongue cancer, leukemia, renal cell carcinoma, gastriccancer, colon adenocarcinoma, endometrial cancer/uterine corpus,cervical cancer/uterine cervix, intrahepatic cholangiocarcinoma,hepatocellular carcinoma, osteosarcoma, urinary bladder carcinoma,malignant melanoma, thyroid cancer, lung adenocarcinoma, prostatecancer, breast cancer, ovarian cancer, pancreatic cancer and glioma. 61.A method of using the nanoemulsion of claim 41, for use in a method forthe treatment and/or prevention of a disease, wherein the methodcomprises administering to a subject the nucleic acid molecule,preferably the disease is selected from the group comprising esophagealcancer, oral squamous cell carcinoma, head and neck cancer, tonguecancer, leukemia, renal cell carcinoma, gastric cancer, colonadenocarcinoma, endometrial cancer/uterine corpus, cervicalcancer/uterine cervix, intrahepatic cholangiocarcinoma, hepatocellularcarcinoma, osteosarcoma, urinary bladder carcinoma, malignant melanoma,thyroid cancer, lung adenocarcinoma, prostate cancer, breast cancer,ovarian cancer, pancreatic cancer and glioma.
 62. A method of using thenanoemulsion of claim 42, for use in a method for the treatment and/orprevention of a disease, wherein the method comprises administering to asubject the nucleic acid molecule, preferably the disease is selectedfrom the group comprising esophageal cancer, oral squamous cellcarcinoma, head and neck cancer, tongue cancer, leukemia, renal cellcarcinoma, gastric cancer, colon adenocarcinoma, endometrialcancer/uterine corpus, cervical cancer/uterine cervix, intrahepaticcholangiocarcinoma, hepatocellular carcinoma, osteosarcoma, urinarybladder carcinoma, malignant melanoma, thyroid cancer, lungadenocarcinoma, prostate cancer, breast cancer, ovarian cancer,pancreatic cancer and glioma.
 63. A method of using the nanoemulsion ofclaim 43, for use in a method for the treatment and/or prevention of adisease, wherein the method comprises administering to a subject thenucleic acid molecule, preferably the disease is selected from the groupcomprising esophageal cancer, oral squamous cell carcinoma, head andneck cancer, tongue cancer, leukemia, renal cell carcinoma, gastriccancer, colon adenocarcinoma, endometrial cancer/uterine corpus,cervical cancer/uterine cervix, intrahepatic cholangiocarcinoma,hepatocellular carcinoma, osteosarcoma, urinary bladder carcinoma,malignant melanoma, thyroid cancer, lung adenocarcinoma, prostatecancer, breast cancer, ovarian cancer, pancreatic cancer and glioma. 64.A method of using the nanoemulsion of claim 44, for use in a method forthe treatment and/or prevention of a disease, wherein the methodcomprises administering to a subject the nucleic acid molecule,preferably the disease is selected from the group comprising esophagealcancer, oral squamous cell carcinoma, head and neck cancer, tonguecancer, leukemia, renal cell carcinoma, gastric cancer, colonadenocarcinoma, endometrial cancer/uterine corpus, cervicalcancer/uterine cervix, intrahepatic cholangiocarcinoma, hepatocellularcarcinoma, osteosarcoma, urinary bladder carcinoma, malignant melanoma,thyroid cancer, lung adenocarcinoma, prostate cancer, breast cancer,ovarian cancer, pancreatic cancer and glioma.
 65. The nanoemulsion ofclaim 33, wherein the method comprises administering a furtherpharmaceutically active agent to the subject.
 66. A method for restoringdrug sensitivity of cancer cells, by administering the microemulsion ofclaim 36, wherein the cancer cells exhibit Notch 1 inducedchemoresistance.
 67. A pharmaceutical composition comprising a nucleicacid molecule of claim 1, and at least one pharmaceutically activeexcipient.
 68. A pharmaceutical composition comprising a nucleic acidmolecule of claim 15, and at least one pharmaceutically activeexcipient.
 69. A pharmaceutical composition comprising a nucleic acidmolecule of claim 16, and at least one pharmaceutically activeexcipient.
 70. A pharmaceutical composition comprising a nucleic acidmolecule of claim 17, and at least one pharmaceutically activeexcipient.
 71. A pharmaceutical composition, wherein said compositioncomprises the nanoemulsion of claim 33 and at least one pharmaceuticallyactive ingredient
 72. A kit comprising a nucleic acid molecule of claim1, and at least use instructions.
 73. A kit comprising a nucleic acidmolecule of claim 15, and at least use instructions.
 74. A kitcomprising a nucleic acid molecule of claim 16, and at least useinstructions.
 75. A kit comprising a nucleic acid molecule of claim 17,and at least use instructions.
 76. A kit comprising a nucleic acidmolecule of claim 18, and at least one use instruction.
 77. A kitcomprising a nucleic acid molecule of claim 19, and at least one useinstruction.
 78. A kit comprising a nanoemulsion of claim 33 and atleast one use instruction.
 79. A kit comprising a nanoemulsion of claim34 and at least one use instruction.
 80. A kit comprising a nanoemulsionof claim 35 and at least one use instruction.